Method for down-regulating IL5 activity

ABSTRACT

The present invention relates to improvements in therapy and prevention of conditions characterized by an elevated level of eosinophil leukocytes, i.e. conditions such as asthma and other chronic allergic diseases. A method is provided for down-regulating interleukin 5 (IL5) by enabling the production of antibodies against IL5 thereby reducing the level of activity of eosinophils. The invention also provides for methods of producing modified IL5 useful in this method as well as for the modified IL5 as such. Also encompassed by the present invention are nucleic acid fragments encoding modified IL5 as well as vectors incorporating these nucleic acid fragments and host cells and cell lines transformed therewith. The invention also provides for a method for the identification of IL5 analogues which are useful in the method of the invention as well as for compositions comprising modified IL5 or comprising nucleic acids encoding the IL5 analogues. The preferred embodiment of the present invention entails the use of variants of IL5, where foreign T helper epitopes are introduced so as to induce production of cross-reactive antibodies capable of binding to autologous IL5.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority under 35 USC 119(e) to U.S. ProvisionalApplication No. 60/132,811 filed May 6, 1999 and is incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to improvements in therapy and preventionof conditions characterized by an elevated level of eosinophilleukocytes, i.e. conditions such as asthma and other chronic allergicdiseases. More specifically, the present invention provides a method fordown-regulating interleukin 5 (IL5) by enabling the production ofantibodies against IL5 thereby reducing the level of activity ofeosinophils. The invention also provides for methods of producingmodified IL5 useful in this method as well as for the modified IL5 assuch. Also encompassed by the present invention are nucleic acidfragments encoding modified IL5 as well as vectors incorporating thesenucleic acid fragments and host cells and cell lines transformedtherewith. The invention also provides for a method for theidentification of IL5 analogues which are useful in the method of theinvention as well as for compositions comprising modified IL5 orcomprising nucleic acids encoding the IL5 analogues.

BACKGROUND OF THE INVENTION

Asthma is a common disease of the airways, affecting about 10% of thepopulation. The present treatments is primarily based on theadministration of steroids and represents a market value exceeding wellover a billion dollars. For yet unknown reasons the incidence andmorbidity of asthmatics have increased worldwide over the past twodecades. Today, an improved understanding of the immunologicalmechanisms involved in asthmatic conditions combined with an explosivedevelopment in biotechnology provides a new basis for the development ofalternative and perhaps better strategies for treatment.

A general feature in the pathogenesis of asthma and other chronicallergic diseases has proven to be elevated numbers of eosinophils,especially in the bronchial mucosa of the lungs. Upon activationeosinophils secrete a number of mediators that are actively involved inthe inflammatory airway response. In the activation of eosinophils,interleukin 5 (IL5) plays an important role.

IL5 is a cytokine found in many mammalian species and among others boththe human and the murine gene for IL5 have been cloned (Tanabe et al.,1987, Campbell et al., 1988). The human gene consists of four exons withthree introns positioned at chromosome 5 and codes for a 134 amino acidresidue precursor, including a 19 amino acid N-terminal leader sequencewhich has the amino acid sequence set forth in SEQ ID NO: 62.Posttranslational cleavage generates the mature 115 amino acid residueprotein (SEQ ID NO: 1). The murine IL5 (mIL5) gene similarly codes for a133 amino acid residue pre-cursor with a 20 amino acid leader sequencewhich has the amino acid sequence set forth in SEQ ID NO: 64. Theprocessed mature mIL5 is thus 113 amino acid residues long (SEQ ID NO:12), missing two N-terminal amino acid residues by alignment with humanIL5. The amino acid sequences of hIL5 and mIL5 are 70% identicalcompared to 77% at nucleotide level of the coding regions (Azuma et al.,1986). Higher similarity was reported within human primates; 99%identity is reported for the coding regions of the human and the Rhesusmonkey nucleotide sequences (Villinger et al., 1995).

The human amino acid sequence has two potential N-glycosylation sitesand the murine three. Human IL5 has been shown to be both N-glycosylatedas well as O-glycosylated at Thr 3. Studies of hIL5 has demonstratedthat the glycosylation is not necessary for the biological activity eventhough the stability seems to be affected by de-glycosylation (Tominagaet al., 1990; Kodama et al., 1993).

Structure of IL5

The active IL5 is a homo-dimer and the 3-dimensional structure ofrecombinant hIL5 has been determined by X-ray crystallography (Milburnet al., 1993). The 2 monomers are organised in an antiparallel mannerand covalently bound by two interchain disulfide bridges (44-87′ and87-44′), thus engaging all 4 cysteines of the 2 monomers.

The secondary structure of the monomers consists of 4 α-helices (A-D)intermitted by 3 linking regions (loops) including two short stretchesof β-sheets. This 4α helix bundle is known as the “common cytokinefold”, which has also been reported for IL-2, IL-4, GM-CSF, and M-CSF.But all these are monomers and the homodimer-structure in which theD-helix completes the 4α helix motif of the opposite monomer is uniqueto IL5.

The native monomers alone has been shown to be biologically inactive(for reviews see Callard & Gearing, 1994; Takutsu et al., 1997). It isnevertheless possible to produce a modified recombinant biologicallyactive monomer by inserting 8 additional amino acid residues in loop 3,connecting the helices C and D. This enables helix D to complete the 4helix structure within one polypeptide chain and thus enable the monomerto interact with its receptor (Dickason & Huston, 1996; Dickason et al.,1996).

The IL5 receptor is primarily present on eosinophils and it is composedof an α-chain and a β-chain. The α-chain of the receptor is specific forIL5 and the β-chain, which assure high-affinity binding and signaltransduction, is shared with the hetero-dimer receptors for IL-3 andGM-CSF. The sharing of a receptor component could be the reason for thecross-competition seen between IL5, IL-3 and GM-CSF (for review, seeLopez et al., 1992). However, it was recently demonstrated that theregulation of the IL5R is distinct from the regulation of the IL-3R andthe GM-CSFR, further indicating a highly specialised role of IL5 in theregulation of the eosinophilic response (Wang et al., 1998).

The C-terminal part of IL5 seems to be important in both binding to theIL5R and for the biological activity, since removal of more than twoC-terminal amino acid residues results in a decline in both the bindingaffinity to the IL5 R and in the biological activity in an IL5 bioassay(Proudfoot et al., 1996). Other residues have also been found to beimportant for binding to the receptor, such as Glu12, which is involvedin binding to the β-chain, while the Arg90 and Glu109 residues areinvolved in the binding to the α-chain of the receptor. In general,binding to the IL5R seems to occur in regions overlapping helices A andD, where helix D is primarily responsible for the binding to thespecific IL5R α-chain (Graber et al., 1995; Takastsu et al., 1997).

IL5's Homology to Other Proteins

The two 4-helix domain motifs seen in the homodimer has strikinglysimilar secondary and tertiary structure as compared to the cytokinefold found in GM-CSF and M-CSF, IL-2, IL-4 and human and porcine growthhormone (Milburn et al., 1993). However, even though strikingsimilarities are also observed in the intron/exon organisation andposition of cysteines (Tanabe et al., 1987; Cambell et al., 1988)suggesting a phylogenetic relationship with IL-2, IL-4 and GM-CSF, nosignificant homology with any of these or other cytokines is observedfrom the amino acid sequence.

Biological Activity of IL5

IL5 is mainly secreted by fully differentiated Th2 cells, mast cells andeosinophils (Cousins et al., 1994; Takutsu et al., 1997). It has beenshown to act on eosinophils, basophiles, cytotoxic T lymphocytes and onmurine B cells (Callard & Gearing, 1994; Takutsu et al., 1997). Theeffects of IL5 on human cells are still a matter of controversy.Augmentation of immunoglobulin synthesis under certain circumstances andbinding to a variety of human B cell lines have been demonstrated. Eventhough mRNA for the hIL5R has been found in human B-cells, the actualpresence of the receptor on these cells has still to be verified(Baumann & Paul, 1997; Huston et al., 1996).

The actions of IL5 on eosinophils include chemotaxis, enhanced adhesionto endothelial cells, activation and terminal differentiation of thecells. Furthermore it has been demonstrated that IL5 prevents matureeosinophils from apoptosis (Yamaguchi et al., 1991). These findings havecontributed to the present concept of IL5 as being the most importantcytokine for eosinophil differentiation (Corrigan & Kay, 1996; Karlen etal., 1998).

Physiologically, IL5 and its associated eosinophil activation isconsidered to serve a protective role against helminthic infections andpossibly against certain tumours, since these diseases are typicallyaccompanied by peripheral blood eosinophilia (Takutsu et al., 1997;Sanderson et al., 1992). It is, however, somewhat speculative as in twostudies the authors failed to show any effect beside eosinophildown-regulating following administration of antibodies against IL5 onthe immunity (e.g. IgE levels) against Nippostrongylus braziliensis orSchistosoma mansoni in mice infected with these parasites (Sher et al.,1990; Coffman et al., 1989).

IL5 Transgenic and “Knock-out” Animals

Studies of transgenic mice expressing IL5 or knock-out mice deficientfor IL5 have given further knowledge of the physiological role of IL5.

Several IL5 transgenic mice have been reported:

A transgenic mouse expressing the IL5 gene in T cells was reported tohave an increased white blood cell level characterised by expansion ofB220+ B lymphocytes and profound eosinophilia. This was accompanied by amassive peritoneal cavity cell exudate dominated by eosinophils andinfiltration of eosinophils in nearly all organ systems (Lee et al.,1997a).

Another transgenic mouse, expressing the IL5 gene under control of ametallothionin promoter was characterised by an increase in the serumlevels of IgM and IgA, a massive eosinophilia in peripheral blood andmany other organs accompanied by the expansion of a distinctive CD5+ Bcell population, which produce auto-antibodies (Tominaga et al., 1991).

A third study involved a transgenic mouse constitutively expressing IL5in the lungs. These animals developed pathophysiological changesresembling those of human asthma, including eosinophil invasion ofperibronchial spaces, epithelial hypertrophy and increased mucusproduction. Furthermore, development of airway hyper responsiveness wasseen in the absence of antigens (Lee et al., 1997b).

IL5-deficient mice (‘knock-out’ mice) have also been studied. These mice(C57BL/6) have no obvious signs of disease and are fertile. Theimmunoglobulin levels and the specific antibody responses to DNP-OVAwere normal. Basal levels of eosinophils are produced, but are 2-3 timeslower than in control animals, indicating that eosinophils can beproduced in the complete absence of IL5. When these mice were infectedwith Mesocestoides corti the eosinophilia normally seen was abolishedand this absence of eosinophilia did not affect the worm burden producedby this parasite (Kopf et al., 1996).

In a study by Foster et al. (1996), the effect of IL5 knock-out on acommon model of atopic airway inflammation was investigated.Sensitisation and aerosol challenge of mice with ovalbumin normallyresult in airway eosinophilia, airway hyperreactivity to β-methacholinand extensive lung damage analogous to that seen in asthma. In the IL5deficient mice the eosinophilia, airway hyperreactivity and lung damagewere abolished. When IL5 expression in these mice was reconstituted, theaero-allergen induced eosinophilia and airway dysfunction were restored.

Pathophysiologic Role of IL5

Asthma affect about 10% of the population worldwide and for yet unknownreasons the incidence and morbidity have increased over the past twodecades (Ortega & Busse, 1997). It is a chronic airway diseasecharacterised by recurrent and usually reversible air flow obstruction,inflammation and hyper responsiveness (Moxam and Costello, 1990). Thisproduces symptoms of wheezing and breathlessness, which in severe casescan be fatal.

The animal experiments referred to above using transgenic miceconstitutively expressing IL5 in the lungs (Lee et al., 1997a) and theIL5 deficient “knock-out” mice (Foster et al., 1996) strongly implicatea crucial role of IL5 in the pathogenesis of asthma. Further evidencesupporting this can be deduced from several studies including asthmaticindividuals.

Eosinophilia has been identified in bronchoalveolar lavage (BAL) fluidand in bronchial mucosal biopsies of subjects with asthma and correlateswith disease severity. Several eosinophil products have been identifiedin the BAL fluid of patients with asthma and numbers of peripheral bloodeosinophils correlate with asthma severity (Ortega & Busse 1997).

IL5 serum concentration was found to be elevated (median concentration150 pg/ml) in 15 out of 29 patients with chronic severe asthma ascompared to control subjects (Alexander et al., 1994).

In another study involving both non-atopic and atopic asthmatics, it wasfound that an enhanced IL5 production by helper T cells seems to causethe eosinophilic inflammation of both atopic and non-atopic asthma (Moriet al., 1997).

Other results also indicate that IL5 has a distinct role in other atopicdiseases. Allergen induced systemic episodes in individuals withallergic rhinitis has recently been shown to correlate to allergeninduced IL5 synthesis rather than IgE (Ohashi et al., 1998). Thecorrelation of atopic reactions is also demonstrated in a study byBarata et al. (1998) in which a significant expression of IL5 by T-cellsin a cutaneous late phase reaction is demonstrated.

These and other results have led several authors as Corrigan & Kay(1996), Danzig & Cuss (1997) to identify and recommend IL5 as a primarytarget in the development of a better treatment for asthma and atopicdiseases involving eosinophilic inflammation. Chronic tissue damaginghypereosinophilia induced by parasitic infection, topical pulmonaryeosinophilia and hypereosinophilic syndrome are examples of otherpathogenic conditions that could be addressed by IL5 down regulation.

In vivo Demonstration of the Role of IL5

In several studies with rodent models of asthma it has been shown thattreatment with monoclonal antibodies against IL5 (anti-IL5 mAb) resultsin dose-related inhibition of eosinophilia, as compared to non-treatedcontrols (Nagai et al., 1993a & b; Chand et al., 1992; Coeffier et al.,1994; Kung et al., 1995; Underwood et al., 1996). In the study by Nagaiet al. (1993a) the effect was also observed by treating the sensitisedBalb/c mice with soluble IL5 receptor α.

In one study with Balb/c mice (Hamelmann et al., 1997) and four studieswith guinea pigs it was additionally shown that anti-IL5 mAb couldinhibit airway hyperreactivity elicited with various substances inantigen sensitised animals (Mauser et al., 1993; Akutsu et al., 1995;van Oosterhout et al., 1995 & 1993). In some of the studies beneficialeffects (cf. table 1) of the anti-IL5 mAb treatment were also observedmicroscopically (Mauser et al., 1993; Akutsu et al., 1995; Kung et al.,1995). Importantly, in the study by Kung et al. (1995) a reduction ofpulmonary inflammation in B6D2F1 mice was seen both when anti-IL5 mAbwas administered hours before antigen challenge and also whenadministered up to five days after antigen challenge, indicating thatthe effect of anti-IL5 mAb may be both prophylactic and therapeutic forairway inflammation. This effect, however, was not observed by Underwoodet al. when guinea pigs were given anti-IL5 mAb two hours after antigenchallenge (Underwood et al., 1996).

In a study using a monkey model of asthma, Mauser et al. (1995) reportedan inhibition of airway hyper reactivity after antigen challenge, whenrat anti mouse-IL5 mAb was given 1 hour before antigen challenge. Inaddition, there was 75% reduction in the number of eosinophils inbronchoalveolar lavage (BAL) of antibody treated animals, as compared tonon-treated controls. The effects on eosinophilia andhyperresponsiveness of anti-IL5 mAb was seen for up to three monthsafter treatment (Mauser et al., 1995). Regarding allergichyperresponsiveness, the results from studies by Nagai et al. (1993a and1993b) document no reduction in hyperresponsiveness in conjunction to areduction of eosinophil numbers in BAL.

All anti-IL5 mAb in vivo experiments mentioned so far have been donewith rat-anti-mouse monoclonal antibodies. Egan et al. (1995) havereported experiments using humanised rat-anti-human IL5 monoclonalantibodies, called Sch 55700. These mAbs, inhibited lung lavageeosinophilia by 75% at a dose of 0,3 mg/kg when administered tosensitised monkeys. When Sch 55700 was given at 1 mg/kg in allergicmice, inhibition of airway eosinophilia was also observed.

Treatment of Asthma at Present and in the Future

The current treatment of asthma is, as mentioned, corticosteroids which,by their anti-inflammatory action, are the most powerful drugs. Besidesthis, β₂ agonists and methyl xanthine derivatives which all causebronchodilation, and disodium chromoglycate which ‘stabilises’ mastcells, thereby preventing mediator release, all have proven beneficialin asthma patients (Ortega & Busse 1997).

Future treatment of asthma may as discussed above include anti-IL5 mAbs.Celltech in corporation with Schering Plough have anti-IL5 mAb in phaseI clinical trial for treatment of asthma. However, treatment withmonoclonal antibodies entails a number of drawbacks. First of all, thedevelopment and production costs for a safe mAB (e.g. a humanised mAB)are very high, resulting in an expensive therapeutic product for the enduser. Second, mABs have the disadvantageous characteristic seen from apatient point of view that they have to be administered with relativelyshort intervals. Third, by nature mABs exhibit a narrow specificityagainst one single epitope of the antigen. And, finally, mABs (evenhumanised) are immunogenic, leading to an increasingly fast inactivationof administered antibodies as treatment progresses over time.

Also use of antisense IL5 oligonucleotides for antisense therapy hasbeen suggested by the company Hybridon for the treatment of asthma,allergies and inflammation. However, the antisense technology has provento be technically difficult and, in fact, conclusive evidence of thefeasibility of antisense therapy in humans has not yet been established.

Finally, WO 97/45448 (Bresagen Limited/Medvet Science) proposes the useof “modified and variant forms of IL5 molecules capable of antagonisingthe activity of IL5” in ameliorating, abating or otherwise reducing theaberrant effects caused by native or mutant forms of IL5. Theantagonizing effect is reported to be the result of the variant forms ofIL5 binding to the low affinity a chain of IL5R but not to the highaffinity receptors; in this way the variants compete with IL5 forbinding to its receptors without exerting the physiological effects ofIL5.

Other atopic diseases involving eosinophilic inflammation are treatedwith either the symptomatica mentioned for asthma or immune therapy (IT)using hyposensitization with allergen extracts. The latter type oftreatment is known to be effective against allergies against one or afew antigens, whereas IT is not feasible in the treatment of multipleallergies. Furthermore, the time scale for obtaining clinicalimprovement in patients susceptible to treatment is very long forconventional IT.

Thus, in spite of existing and possible future therapies for chronicallergic diseases such as asthma, there is a definite need foralternative ways of treating and ameliorating this and other chronicallergic diseases.

OBJECT OF THE INVENTION

The object of the present invention is to provide novel therapiesagainst chronic allergic conditions (such as asthma) characterized byeosinophilia. A further object is to develop an autovaccine against IL5,in order to obtain a novel treatment for asthma and for otherpathological disorders involving chronic airway inflammation.

SUMMARY OF THE INVENTION

The T-cell derived cytokine IL5 has, as mentioned above, a crucial rolein orchestrating the eosinophilic response, affecting both theproduction, the localisation and the activation of eosinophils. As IL5has not otherwise been reported to have a central role in thedevelopment of a protective immune response, this particular cytokine isin the opinion of the inventors an attractive therapeutic target for thetreatment of asthma.

The general aim according to the present invention is to decrease thepathogenic levels of eosinophils in the airways of the asthma patient bydown-regulating of the IL5 levels, since eosinophils depend on IL5 forattraction and activation. The result of a decreased eosinophil numberin the airway mucosa would be a concomitant decrease in the airwayinflammation, corresponding to a clinical improvement in the asthmaticpatient.

The potential effect of such an approach has already been demonstratedin studies using anti IL5 monoclonal antibodies in animal models ofairway inflammation, cf. the “PREAMBLE TO EXAMPLES”.

This current invention, however, takes the results obtained throughpassive immunisation one step further by using the approach ofgenerating an active immune response through the concept ofautovaccination. To the best of the inventor's knowledge, such anapproach has never been suggested before.

The advantage of treating asthmatics with an IL5 autovaccine, ascompared to current treatment with corticosteroids etc., is a reductionand/or elimination of side effects and most likely a better effect interms of duration. When compared to anti-IL5 mAbs, the effect of aninduced polyclonal Ab response is expected to be superior to passivelyinjected monoclonal immunoglobulins since the polyclonal response has abroader specificity. Improvements with respect to administration regimenare also expected (since effective autovaccines described hereintypically would require a maximum of 2-6 administrations per year).

When compared to hyposensitization, the present invention offers theattractive aspect of being non-specific; this is especially relevantwhen dealing with multi-allergic patients.

Thus, in its broadest and most general scope, the present inventionrelates to a method for in vivo down-regulation of interleukin 5 (IL5)activity in an animal, including a human being, the method comprisingeffecting presentation to the animal's immune system of animmunologically effective amount of

at least one IL5 polypeptide or subsequence thereof which has beenformulated so that immunization of the animal with the IL5 polypeptideor subsequence thereof induces production of antibodies against the IL5polypeptide, and/or

at least one IL5 analogue wherein is introduced at least onemodification in the IL5 amino acid sequence which has as a result thatimmunization of the animal with the analogue induces production ofantibodies against the IL5 polypeptide.

The most attractive aspect of this approach is that e.g. asthma can becontrolled by periodic but not very frequent immunizations, in contrastto a therapeutic approach which involves administration of anti-IL5 ormolecules having a binding affinity to IL5 analogous therewith. It isexpected that 1-4 annual injections with an immunogenic compositionaccording to the invention will be sufficient to obtain the desiredeffect, whereas administration of other inhibitors of IL5 activity doesor will require daily, or at least weekly, administrations.

The invention also relates to IL5 analogues as well as to nucleic acidfragments encoding a subset of these. Also immunogenic compositionscomprising the analogues or the nucleic acid fragments are part of theinvention.

The invention also relates to a method of identifying analogues of IL5as well as a method for preparing a composition comprising the IL5analogues.

LEGENDS TO THE FIGURES

FIG. 1: The amino acid sequence of the mature human IL5 (SEQ ID NO: 1).The aligned murine sequence is included (SEQ ID NO: 12), but onlypositions that differ from the human sequence are displayed. The two“*”s indicate the missing N-terminal residues of the murine IL5. TheN-glycosylation positions are marked with double underlining, theO-glycosylated threonines of human IL5 are given in italics, and thecysteines in bold.

FIG. 2: The dimer and monomer structures of human IL5.

A: Dimer structure of hIL5. The structure has only been obtained forresidues 5-112, which means that the O-glycosylation site at Thr3 is notincluded.

B: The same structure as in A, with the assignment of the helices (A-Dand A′-D′).

C: The monomer hIL5 with the amino acid residues differing from the mIL5shown in light grey.

FIG. 3: The aligned mature human IL5 (hIL5) and murine IL5 (mIL5) aminoacid sequences (SEQ ID NOs: 1 and 12) with indications of suitablesubstitution regions. The 4 α-helices A-D are surrounded by solid-lineboxes, the β-sheets are double underlined and the positions of the twocysteines are marked with “▾”. Identical residues in the two sequencesare marked with “−” and non-identical residues with “*”. Loop 1 spansbetween helices A and B, Loop 2 spans between helices B and C, and loop3 spans between loops C and D. Amino acid sequences to be substitutedwith foreign T_(H) epitope containing peptides are marked in bold; onesuch sequence is surrounded by a dot-lined box because of residuesoverlapping with those substituted in a different construct. The aminoacid sequences of 10 constructs (5 derived from human and 5 derived frommurine IL5) are set forth in SEQ ID NOs: 2-11 and 13-22.

FIG. 4: ELISA results of DNA immunization testing two mIL5 autovaccineDNA vaccines.

Mice were DNA vaccinated with naked plasmid DNA encoding eitherovalbumin, mIL5wt, mIL5.1 or mIL5.5. Sera obtained at day 77 were testedfor reactivity against ovalbumin and murine IL5. Polystyrene microtiterplates (Maxisorp, Nunc) were coated with ovalbumin (1 μg/well, Sigma) orpurified recombinant murine IL5 (0.1 μg/well, E1320). The reactivitiesof diluted sera added to the wells were visualised using a goatanti-mouse secondary antibody. OD490 readings of the pre-bleeds weresubtracted from the OD490 readings of the test samples, and theresulting values were presented for each individual mouse as bars. TheOD490 readings of the pre-bleeds (in 1:25 dilution) were ranging from0.025-0.034. Crucifixes indicate dead animals.

FIG. 5: Schematic representation of murine IL5 based autovaccineconstructs.

The top figure represents murine wild-type IL5 monomer with helices A-C,loops 1-3 and the flexible C-terminal region. Remaining figuresrepresent different autovaccine constructs having in-substitutions ofthe tetanus toxoid epitopes P2 and P30 in various positions. Specificconstructs are detailed in the Examples.

DETAILED DISCLOSURE OF THE INVENTION Definitions

In the following, a number of terms used in the present specificationand claims will be defined and explained in detail in order to clarifythe metes and bounds of the invention.

The terms “T-lymphocyte” and “T-cell” will be used interchangeably forlymphocytes of thymic origin which are responsible for various cellmediated immune responses as well as for helper activity in the humeralimmune response. Likewise, the terms “B-lymphocyte” and “B-cell” will beused interchangeably for antibody-producing lymphocytes.

An “IL5 polypeptide” is herein intended to denote polypeptides havingthe amino acid sequence of the above-discussed IL5 proteins derived fromhumans and mice (or truncates thereof sharing a substantial amount ofB-cell epitopes with intact IL5), but also polypeptides having the aminoacid sequence identical to xeno-analogues of these two proteins isolatedfrom other species are embraced by the term. Also unglycosylated formsof IL5 which are prepared in prokaryotic system are included within theboundaries of the term as are forms having varying glycosylationpatterns due to the use of e.g. yeasts or other non-mammalian eukaryoticexpression systems. It should, however, be noted that when using theterm “an IL5 polypeptide” it is intended that the polypeptide inquestion is normally non-immunogenic when presented to the animal to betreated. In other words, the IL5 polypeptide is a self-protein or is axeno-analogue of such a self-protein which will not normally give riseto an immune response against IL5 of the animal in question.

An “IL5 analogue” is an IL5 polypeptide which has been subjected tochanges in its primary structure. Such a change can e.g. be in the formof fusion of an IL5 polypeptide to a suitable fusion partner (i.e. achange in primary structure exclusively involving C- and/or N-terminaladditions of amino acid residues) and/or it can be in the form ofinsertions and/or deletions and/or substitutions in the IL5polypeptide's amino acid'sequence. Also encompassed by the term arederivatized IL5 molecules, cf. the discussion below of modifications ofIL5.

It should be noted that the use as a vaccine in a human of e.g. a canineanalogue of human IL5 can be imagined to produce the desired immunityagainst IL5. Such use of an xeno-analogue for immunization is alsoconsidered to be an “IL5 analogue” as defined above.

When using the abbreviation “IL5” herein, this is intended as areference to the amino acid sequence of mature, wildtype IL5 (alsodenoted “IL5m” and “IL5wt” herein). Mature human IL5 is denoted hIL5,hIL5m or hIL5wt, and murine mature IL5 is denoted mIL5, mIL5m, ormIL5wt. In cases where a DNA construct includes information encoding aleader sequence or other material, this will normally be clear from thecontext.

The term “polypeptide” is in the present context intended to mean bothshort peptides of from 2 to 10 amino acid residues, oligopeptides offrom 11 to 100 amino acid residues, and polypeptides of more than 100amino acid residues. Furthermore, the term is also intended to includeproteins, i.e. functional biomolecules comprising at least onepolypeptide; when comprising at least two polypeptides, these may formcomplexes, be covalently linked, or may be non-covalently linked. Thepolypeptide(s) in a protein can be glycosylated and/or lipidated and/orcomprise prosthetic groups.

The term “subsequence” means any consecutive stretch of at least 3 aminoacids or, when relevant, of at least 3 nucleotides, derived directlyfrom a naturally occurring IL5 amino acid sequence or nucleic acidsequence, respectively.

The term “animal” is in the present context in general intended todenote an animal species (preferably mammalian), such as Homo sapiens,Canis domesticus, etc. and not just one single animal. However., theterm also denotes a population of such an animal species, since it isimportant that the individuals immunized according to the method of theinvention all harbour substantially the same IL5 allowing forimmunization of the animals with the same immunogen(s). If, forinstance, genetic variants of IL5 exists in different human populationit may be necessary to use different immunogens in these differentpopulations in order to be able to break the autotolerance towards IL5in each population. It will be clear to the skilled person that ananimal in the present context is a living being which has an immunesystem. It is preferred that the animal is a vertebrate, such as amammal.

By the term “in vivo down-regulation of IL5 activity” is herein meantreduction in the living organism of the number of interactions betweenIL5 and its receptors (or between IL5 and other possible biologicallyimportant binding partners for this molecule). The down-regulation canbe obtained by means of several mechanisms: Of these, simpleinterference with the active site in IL5 by antibody binding is the mostsimple. However, it is also within the scope of the present inventionthat the antibody binding results in removal of IL5 by scavenger cells(such as macrophages and other phagocytic cells).

The expression “effecting presentation . . . to the immune system” isintended to denote that the animal's immune system is subjected to animmunogenic challenge in a controlled manner. As will appear from thedisclosure below, such challenge of the immune system can be effected ina number of ways of which the most important are vaccination withpolypeptide containing “pharmaccines” (i.e. a vaccine which isadministered to treat or ameliorate ongoing disease) or nucleic acid“pharmaccine” vaccination. The important result to achieve is thatimmune competent cells in the animal are confronted with the antigen inan immunologically effective manner, whereas the precise mode ofachieving this result is of less importance to the inventive ideaunderlying the present invention.

The term “immunogenically effective amount” has its usual meaning in theart, i.e. an amount of an immunogen which is capable of inducing animmune response which significantly engages pathogenic agents whichshare immunological features with the immunogen.

When using the expression that the IL5 has been “modified” is hereinmeant a chemical modification of the polypeptide which constitutes thebackbone of IL5. Such a modification can e.g. be derivatization (e.g.alkylation, acylation, esterification etc.) of certain amino acidresidues in the IL5 sequence, but as will be appreciated from thedisclosure below, the preferred modifications comprise changes of (oradditions to) the primary structure of the IL5 amino acid sequence.

When discussing “autotolerance towards IL5” it is understood that sinceIL5 is a self-protein in the population to be vaccinated, normalindividuals in the population do not mount an immune response againstIL5; it cannot be excluded, though, that occasional individuals in ananimal population might be able to produce antibodies against; nativeIL5, e.g. as part of an autoimmune disorder. At any rate, an animal willnormally only be autotolerant towards its own IL5, but it cannot beexcluded that IL5 analogues derived from other animal species or from apopulation having a different IL5 phenotype would also be tolerated bysaid animal.

A “foreign T-cell epitope” (or: “foreign T-lymphocyte epitope”) is apeptide which is able to bind to an MHC molecule and which stimulatesT-cells in an animal species. Preferred foreign T-cell epitopes in theinvention are “promiscuous” epitopes, i.e. epitopes which bind to asubstantial fraction of a particular class of MHC molecules in an animalspecies or population. Only a very limited number of such promiscuousT-cell epitopes are known, and they will be discussed in detail below.It should be noted that in order for the immunogens which are usedaccording to the present invention to be effective in as large afraction of an animal population as possible, it may be necessary to 1)insert several foreign T-cell epitopes in the same IL5 analogue or 2)prepare several IL5 analogues wherein each analogue has a differentpromiscuous epitope inserted. It should be noted also that the conceptof foreign T-cell epitopes also encompasses use of cryptic T-cellepitopes, i.e. epitopes which are derived from a self-protein and whichonly exerts immunogenic behaviour when existing in isolated form withoutbeing part of the self-protein in question.

A “foreign T helper lymphocyte epitope” (a foreign T_(H) epitope) is aforeign T cell epitope which binds an MHC Class II molecule and can bepresented on the surface of an antigen presenting cell (APC) bound tothe MHC Class II molecule.

A “functional part” of a (bio)molecule is in the present contextintended to mean the part of the molecule which is responsible for atleast one of the biochemical or physiological effects exerted by themolecule. It is well-known in the art that many enzymes and othereffector molecules have an active site which is responsible for theeffects exerted by the molecule in question. Other parts of the moleculemay serve a stabilizing or solubility enhancing purpose and cantherefore be left out if these purposes are not of relevance in thecontext of a certain embodiment of the present invention. For instanceit is possible to use certain other cytokines as a modifying moiety inIL5 (cf. the detailed discussion below), and in such a case, the issueof stability may be irrelevant since the coupling to IL5 provides thestability necessary.

The term “adjuvant” has its usual meaning in the art of vaccinetechnology, i.e. a substance or a composition of matter which is 1) notin itself capable of mounting a specific immune response against theimmunogen of the vaccine, but which is 2) nevertheless capable ofenhancing the immune response against the immunogen. Or, in other words,vaccination with the adjuvant alone does not provide an immune responseagainst the immunogen, vaccination with the immunogen may or may notgive rise to an immune response against the immunogen, but thecombination of vaccination with immunogen and adjuvant induces an immuneresponse against the immunogen which is stronger than that induced bythe immunogen alone.

“Targeting” of a molecule is in the present context intended to denotethe situation where a molecule upon introduction in the animal willappear preferentially in certain tissue(s) or will be preferentiallyassociated with certain cells or cell types. The effect can beaccomplished in a number of ways including formulation of the moleculein composition facilitating targeting or by introduction in the moleculeof groups which facilitates targeting. These issues will be discussed indetail below.

“Stimulation of the immune system” means that a substance or compositionof matter exhibits a general, non-specific immunostimulatory effect. Anumber of adjuvants and putative adjuvants (such as certain cytokines)share the ability to stimulate the immune system. The result of using animmunostimulating agent is an increased “alertness” of the immune systemmeaning that simultaneous or subsequent immunization with an immunogeninduces a significantly more effective immune response compared toisolated use of the immunogen

Preferred Embodiments of IL5 Activity Down-regulation

It is preferred that the IL5 polypeptide used as an immunogen in themethod of the invention is a modified molecule wherein at least onechange is present in the IL5 amino acid sequence, since the chances ofobtaining the all-important breaking of autotolerance towards IL5 isgreatly facilitated that way. It should be noted that this does notexclude the possibility of using such a modified IL5 in formulationswhich further facilitate the breaking of autotolerance against IL5, e.g.formulations containing certain adjuvants discussed in detail below.

It has been shown (in Dalum I et al., 1996, J. Immunol. 157: 4796-4804)that potentially self-reactive B-lymphocytes recognizing self-proteinsare physiologically present in normal individuals. However, in order forthese B-lymphocytes to be induced to actually produce antibodiesreactive with the relevant self-proteins, assistance is needed fromcytokine producing T-helper lymphocytes (T_(H)-cells orT_(H)-lymphocytes). Normally this help is not provided becauseT-lymphocytes in general do not recognize T-cell epitopes derived fromself-proteins when presented by antigen presenting cells (APCs).However, by providing an element of “foreignness” in a self-protein(i.e. by introducing an immunologically significant modification),T-cells recognizing the foreign element are activated upon recognizingthe foreign epitope on an APC (such as, initially, a mononuclear cell).Polyclonal B-lymphocytes (which are also specialised APCs) capable ofrecognising self-epitopes on the modified self-protein also internalisethe antigen and subsequently presents the foreign T-cell epitope(s)thereof, and the activated T-lymphocytes subsequently provide cytokinehelp to these self-reactive polyclonal B-lymphocytes. Since theantibodies produced by these polyclonal B-lymphocytes are reactive withdifferent epitopes on the modified polypeptide, including those whichare also present in the native polypeptide, an antibody cross-reactivewith the non-modified self-protein is induced. In conclusion, theT-lymphocytes can be led to act as if the population of polyclonalB-lymphocytes have recognised an entirely foreign antigen, whereas infact only the inserted epitope(s) is/are foreign to the host. In thisway, antibodies capable of cross-reacting with non-modifiedself-antigens are induced.

Several ways of modifying a peptide self-antigen in order to obtainbreaking of autotolerance are known in the art. Hence, according to theinvention, the modification can include that

at least one foreign T-cell epitope is introduced, and/or

at least one first moiety is introduced which effects targeting of themodified molecule to an antigen presenting cell (APC), and/or

at least one second moiety is introduced which stimulates the immunesystem, and/or

at least one third moiety is introduced which optimises presentation ofthe modified IL5 polypeptide to the immune system.

However, all these modifications should be carried out while maintaininga substantial fraction of the original B-lymphocyte epitopes in IL5,since the B-lymphocyte recognition of the native molecule is therebyenhanced.

In one preferred embodiment, side groups (in the form of foreign T-cellepitopes or the above-mentioned first, second and third moieties) arecovalently or non-covalently introduced. This is intended to mean thatstretches of amino acid residues derived from IL5 are derivatizedwithout altering the primary amino acid sequence, or at least withoutintroducing changes in the peptide bonds between the individual aminoacids in the chain.

An alternative, and preferred, embodiment utilises amino acidsubstitution and/or deletion and/or insertion and/or addition (which maybe effected by recombinant means or by means of peptide synthesis;modifications which involves longer stretches of amino acids can giverise to fusion polypeptides). One especially preferred version of thisembodiment is the technique described in WO 95/05849, which discloses amethod for down-regulating self-proteins by immunising with analogues ofthe self-proteins wherein a number of amino acid sequence(s) has beensubstituted with a corresponding number of amino acid sequence(s) whicheach comprise a foreign immunodominant T-cell epitope, while at the sametime maintaining the overall tertiary structure of the self-protein inthe analogue. For the purposes of the present invention, it is howeversufficient if the modification (be it an amino acid insertion, addition,deletion or substitution) gives rise to a foreign T-cell epitope and atthe same time preserves a substantial number of the B-cell epitopes inIL5. However, in order to obtain maximum efficacy of the immune responseinduced, it is preferred that the overall tertiary structure of IL5 ismaintained in the modified molecule.

The following formula describes the IL5 constructs generally covered bythe invention:

(MOD₁)_(s1)(IL5_(e1))_(n1)(MOD₂)_(s2)(IL5_(e) ₂)_(n2). . .(MOD_(x))_(sx)(IL5_(ex))_(nx)  (I)

where IL5_(e1)-IL5_(ex) are x B-cell epitope containing subsequences ofIL5 which independently are identical or non-identical and which maycontain or not contain foreign side groups, x is an integer ≧3, n1-nxare x integers ≧0 (at least one is ≧1), MOD₁-MOD_(x) are x modificationsintroduced between the preserved B-cell epitopes, and s₁-s_(x) are xintegers ≧0 (at least one is ≧1 if no side groups are introduced in theIL5_(e) sequences). Thus, given the general functional restraints on theimmunogenicity of the constructs, the invention allows for all kinds ofpermutations of the original IL5 sequence, and all kinds ofmodifications therein. Thus, included in the invention are modified IL5obtained by omission of parts of the IL5 sequence which e.g. exhibitadverse effects in vivo or omission of parts which could give rise toundesired immunological reactions.

Maintenance of a substantial fraction of B-cell epitopes or even theoverall tertiary structure of a protein which is subjected tomodification as described herein can be achieved in several ways. One issimply to prepare a polyclonal antiserum directed against IL5 (e.g. anantiserum prepared in a rabbit) and thereafter use this antiserum as atest reagent (e.g. in a competitive ELISA) against the modified proteinswhich are produced. Modified versions (analogues) which react to thesame extent with the antiserum as does IL5 must be regarded as havingthe same overall tertiary structure as IL5 whereas analogues exhibitinga limited (but still significant and specific) reactivity with such anantiserum are regarded as having maintained a substantial fraction ofthe original B-cell epitopes.

Alternatively, a selection of monoclonal antibodies reactive withdistinct epitopes on IL5 can be prepared and used as a test panel. Thisapproach has the advantage of allowing 1) an epitope mapping of IL5 and2) a mapping of the epitopes which are maintained in the analoguesprepared.

Of course, a third approach would be to resolve the 3-dimensionalstructure of IL5 or of a biologically active truncate thereof (cf.above) and compare this to the resolved three-dimensional structure ofthe analogues prepared. Three-dimensional structure can be resolved bythe aid of X-ray diffraction studies and NMR-spectroscopy. Furtherinformation relating to the tertiary structure can to some extent beobtained from circular dichroism studies which have the advantage ofmerely requiring the polypeptide in pure form (whereas X-ray diffractionrequires the provision of crystallized polypeptide and NMR requires theprovision of isotopic variants of the polypeptide) in order to provideuseful information about the 25 tertiary structure of a given molecule.However, ultimately X-ray diffraction and/or NMR are necessary to obtainconclusive data since circular dichroism can only provide indirectevidence of correct 3-dimensional structure via information of secondarystructure elements.

One preferred embodiment of the invention utilises multiplepresentations of B-lymphocyte epitopes of IL5 (i.e. formula I wherein atleast one B-cell epitope is present in two positions). This effect canbe achieved in various ways, e.g. by simply preparing fusionpolypeptides comprising the structure (IL5)_(m), where m is an integer≧2 and then introduce the modifications discussed herein in at least oneof the IL5 sequences, or alternatively, inserted between at least two ofthe IL5 amino acid sequences. It is preferred that the modificationsintroduced includes at least one duplication of a B-lymphocyte epitopeand/or the introduction of a hapten.

As mentioned above, the introduction of a foreign T-cell epitope can beaccomplished by introduction of at least one amino acid insertion,addition, deletion, or substitution. Of course, the normal situationwill be the introduction of more than one change in the amino acidsequence (e.g. insertion of or substitution by a complete T-cellepitope) but the important goal to reach is that the IL5 analogue, whenprocessed by an antigen presenting cell (APC), will give rise to such aforeign immunodominant T-cell epitope being presented in context of anMCH Class II molecule on the surface of the APC. Thus, if the IL5 aminoacid sequence in appropriate positions comprises a number of amino acidresidues which can also be found in a foreign T_(H) epitope then theintroduction of a foreign T_(H) epitope can be accomplished by providingthe remaining amino acids of the foreign epitope by means of amino acidinsertion, addition, deletion and substitution. In other words, it isnot necessary to introduce a complete T_(H) epitope by insertion orsubstitution.

It is preferred that the number of amino acid insertions, deletions,substitutions or additions is at least 2, such as 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 25 insertions,substitutions, additions or deletions. It is furthermore preferred thatthe number of amino acid insertions, substitutions, additions ordeletions is not in excess 30 of 150, such as at most 100, at most 90,at most 80, and at most 70. It is especially preferred that the numberof substitutions, insertions, deletions, or additions does not exceed60, and in particular the number should not exceed 50 or even 40. Mostpreferred is a number of not more than 30. With respect to amino acidadditions, it should be noted that these, when the resulting constructis in the form of a fusion polypeptide, is often considerably higherthan 150.

Preferred embodiments of the invention includes modification byintroducing at least one foreign immunodominant T_(H) epitope. It willbe understood that the question of immune dominance of a T_(H) epitopedepends on the animal species in question. As used herein, the term“immunodominance” simply refers to epitopes which in the vaccinatedindividual gives rise to a significant immune response, but it is awell-known fact that a T_(H) epitope which is immunodominant in oneindividual is not necessarily immunodominant in another individual ofthe same species, even though it may be capable of binding MHC-IImolecules in the latter individual.

Another important point is the issue of MHC restriction of T_(H)epitopes. In general, naturally occurring T_(H) epitopes are MHCrestricted, i.e. a certain peptide constituting a T_(H) epitope willonly bind effectively to a subset of MHC Class II molecules. This inturn has the effect that in most cases the use of one specific T_(H)epitope will result in a vaccine component which is effective in afraction of the population only, and depending on the size of thatfraction, it can be necessary to include more T_(H) epitopes in the samemolecule, or alternatively prepare a multi-component vaccine wherein thecomponents are IL5 variants which are distinguished from each other bythe nature of the T_(H) epitope introduced.

If the MHC restriction of the T-cells used is completely unknown (forinstance in a situation where the vaccinated animal has a poorly definedMHC composition), the fraction of the animal population covered by aspecific vaccine composition can be determined by means of the followingformula: $\begin{matrix}{f_{population} = {1 - {\prod\limits_{i = 1}^{n}\quad \left( {1 - p_{i}} \right)}}} & ({II})\end{matrix}$

where p_(i) is the frequency in the population of responders to thei^(th) foreign T-cell epitope present in the vaccine composition, and nis the total number of foreign T-cell epitopes in the vaccinecomposition. Thus, a vaccine composition containing 3 foreign T-cellepitopes having response frequencies in the population of 0.8, 0.7, and0.6, respectively, would give

1−0.2×0.3×0.4=0.976

i.e. 97.6 percent of the population will statistically mount an MHC-IImediated response to the vaccine.

The above formula does not apply in situations where a more or lessprecise MHC restriction pattern of the peptides used is known. If, forinstance a certain peptide only binds the human MHC-II molecules encodedby HLA-DR alleles DR1, DR3, DR5, and DR7, then the use of this peptidetogether with another peptide which binds the remaining MHC-II moleculesencoded by HLA-DR alleles will accomplish 100% coverage in thepopulation in question. Likewise, if the second peptide only binds DR3and DR5, the addition of this peptide will not increase the coverage atall. If one bases the calculation of population response purely on MHCrestriction of T-cell epitopes in the vaccine, the fraction of thepopulation covered by a specific vaccine composition can be determinedby means of the following formula: $\begin{matrix}{f_{population} = {1 - {\prod\limits_{i = 1}^{3}\quad \left( {1 - \phi_{j}} \right)^{2}}}} & ({III})\end{matrix}$

wherein φ_(j) is the sum of frequencies in the population of allelichaplotypes encoding MHC molecules which bind any one of the T-cellepitopes in the vaccine and which belong to the j^(th) of the 3 knownHLA loci (DP, DR and DQ); in practice, it is first determined which MHCmolecules will recognize each T-cell epitope in the vaccine andthereafter these MHC molecules are listed by type (DP, DR and DQ)—then,the individual frequencies of the different listed allelic haplotypesare summed for each type, thereby yielding φ₁, φ₂, and φ₃.

It may occur that the value p_(i) in formula II exceeds thecorresponding theoretical value π_(i): $\begin{matrix}{\pi_{i} = {1 - {\prod\limits_{j = 1}^{3}\quad \left( {1 - v_{j}} \right)^{2}}}} & ({IV})\end{matrix}$

wherein V_(j) is the sum of frequencies in the population of allelichaplotypes encoding MHC molecules which bind the i^(th) T-cell epitopein the vaccine and which belong to the j^(th) of the 3 known HLA loci(DP, DR and DQ). This means that in 1−π_(i) of the population there is afrequency of responders of f_(residual) _(—)_(i)=(p_(i)−π_(i))/(1−π_(i)). Therefore, formula III can be adjusted soas to yield formula V: $\begin{matrix}{f_{population} = {1 - {\prod\limits_{j = 1}^{3}\quad \left( {1 - \phi_{j}} \right)^{2}} + \left( {1 - {\prod\limits_{i = 1}^{n}\quad \left( {1 - f_{residual\_ i}} \right)}} \right)}} & (V)\end{matrix}$

where the term 1-f_(residual) _(—) _(i) is set to zero if negative. Itshould be noted that formula V requires that all epitopes have beenhaplotype mapped against identical sets of haplotypes.

Therefore, when selecting T-cell epitopes to be introduced in the IL5analogue, it is important: to include all knowledge of the epitopeswhich is available: 1) The frequency of responders in the population toeach epitope, 2) MHC restriction data, and 3) frequency in thepopulation of the relevant haplotypes.

There exists a number of naturally occurring “promiscuous” T-cellepitopes which are active in a large proportion of individuals of ananimal species or an animal population and these are preferablyintroduced in the vaccine, thereby reducing the need for a very largenumber of different IL5 analogues in the same vaccine.

The promiscuous epitope can according to the invention be a naturallyoccurring human T-cell epitope such as epitopes from tetanus toxoid(e.g. the P2 and P30 epitopes), diphtheria toxoid, Influenza virushemagluttinin (HA), and P. falciparum CS antigen.

Over the years a number of other promiscuous T-cell epitopes have beenidentified. Especially peptides capable of binding a large proportion ofHLA-DR molecules encoded by the different HLA-DR alleles have beenidentified and these are all possible T-cell epitopes to be introducedin the IL5 analogues used according to the present invention. Cf. alsothe epitopes discussed in the following references which are hereby allincorporated by reference herein: WO 98/23635 (Frazer IH et al.,assigned to The University of Queensland); Southwood S et. al, 1998, J.Immunol. 160: 3363-3373. Sinigaglia F et al., 1988, Nature 336: 778-780;Chicz RM et al., 1993, J. Exp. Med 178: 27-47; Hammer J et al., 1993,Cell 74: 197-203; and Falk K et al., 1994, Immunogenetics 39: 230-242.The latter reference also deals with HLA-DQ and -DP ligands. Allepitopes listed in these 5 references are relevant as candidate naturalepitopes to be used in the present invention, as are epitopes whichshare common motifs with these.

Alternatively, the epitope can be any artificial T-cell epitope which iscapable of binding a large proportion of MHC Class II molecules. In thiscontext the pan DR epitope peptides (“PADRE”) described in WO 95/07707and in the corresponding paper Alexander J et al., 1994, Immunity 1:751-761 (both disclosures are incorporated by reference herein) areinteresting candidates for epitopes to be used according to the presentinvention. It should be noted that the most effective PADRE peptidesdisclosed in these papers carry D-amino acids in the C- and N-termini inorder to improve stability when administered. However, the presentinvention primarily aims at incorporating the relevant epitopes as partof the modified IL5 which should then subsequently be broken downenzymatically inside the lysosomal compartment of APCs to allowsubsequent presentation in the context of an MHC-II molecule andtherefore it is not expedient to incorporate D-amino acids in theepitopes used in the present invention.

One especially preferred PADRE peptide is the one having the amino acidsequence AKFVAAWTLKAAA (SEQ ID NO: 65) or an immunologically effectivesubsequence thereof. This, and other epitopes having the same lack ofMHC restriction are preferred T-cell epitopes which should be present inthe IL5 analogues used in the inventive method. Such super-promiscuousepitopes will allow for the most simple embodiments of the inventionwherein only one single modified IL5 is presented to the vaccinatedanimal's immune system.

As mentioned above, the modification of IL5 can also include theintroduction of a first moiety which targets the modified IL5 to an APCor a B-lymphocyte. For instance, the first moiety can be a specificbinding partner for a B-lymphocyte specific surface antigen or for anAPC specific surface antigen. Many such specific surface antigens areknown in the art. For instance, the moiety can be a carbohydrate forwhich there is a receptor on the B-lymphocyte or on the APC (e.g. mannanor mannose). Alternatively, the second moiety can be a hapten. Also anantibody fragment which specifically recognizes a surface molecule onAPCs or lymphocytes can be used as a first moiety (the surface moleculecan e.g. be an FCγ receptor of macrophages and monocytes, such as FCγRIor, alternatively any other specific surface marker such as CD40 orCTLA-4). It should be noted that all these exemplary targeting moleculescan be used as part of an adjuvant also, cf. below.

As an alternative or supplement to targeting the modified IL5polypeptide to a certain cell type in order to achieve an enhancedimmune response, it is possible to increase the level of responsivenessof the immune system by including the abovementioned second moiety whichstimulates the immune system. Typical examples of such second moietiesare cytokines, and heat-shock proteins or molecular chaperones, as wellas effective parts thereof.

Suitable cytokines to be used according to the invention are those whichwill normally also function as adjuvants in a vaccine composition, i.e.for instance interferon γ (IFN-γ), Flt3L, interleukin 1 (IL-1),interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6),interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15),and granulocyte-macrophage colony stimulating factor (GM-CSF);alternatively, the functional part of the cytokine molecule may sufficeas the second moiety. With respect to the use of such cytokines asadjuvant substances, cf. the discussion below. It should be noted thatuse of both IL-4 and IL-13 should be exercised very carefully, if atall, as both molecules are known as key effector molecules in thepathophysiology of atopy and asthma.

According to the invention, suitable heat-shock proteins or molecularchaperones used as the second moiety can be HSP70, HSP90, HSC70, GRP94(also known as gp96, cf. Wearsch PA et al. 1998, Biochemistry 37:5709-19), and CRT (calreticulin).

Alternatively, the second moiety can be a toxin, such as listeriolycin(LLO), lipid A and heat-labile enterotoxin. Also, a number ofmycobacterial derivatives such as MDP (muramyl dipeptide) and thetrehalose diesters TDM and TDE are interesting possibilities.

Also the possibility of introducing a third moiety which enhances thepresentation of the modified IL5 to the immune system is an importantembodiment of the invention. The art has shown several examples of thisprinciple. For instance, it is known that the palmitoyl lipidationanchor in the Borrelia burgdorferi protein OspA can be utilised so as toprovide self-adjuvating polypeptides (cf. e.g. WO 96/40718). It seemsthat the lipidated proteins form up micelle-like structures with a coreconsisting of the lipidation anchor parts of the polypeptides and theremaining parts of the molecule protruding therefrom, resulting inmultiple presentations of the antigenic determinants. Hence, the use ofthis and related approaches using different lipidatibn anchors (e.g. amyristyl group, a myristyl group, a farnesyl group, a geranyl-geranylgroup, a GPI-anchor, and an N-acyl diglyceride group) are preferredembodiments of the invention, especially since the provision of such alipidation anchor in a recombinantly produced protein is fairlystraightforward and merely requires use of e.g. a naturally occurringsignal 'sequence as a fusion partner for the modified IL5 polypeptide.Another possibility is use of the C3d fragment of complement factor C3or C3 itself (cf. Dempsey et al., 1996, Science 271, 348-350 and Lou &Kohler, 1998, Nature Biotechnology 16, 458-462).

An alternative embodiment of the invention which also results in thepreferred presentation of multiple (e.g. at least 2) copies of theimportant epitopic regions of IL5 to the immune system is the covalentor non-covalent coupling of IL5, subsequence or variants thereof tocertain carrier molecules. For instance, polymers can be used, e.g.carbohydrates such as dextran, cf. e.g. Lees A et al., 1994, Vaccine 12:1160-1166; Lees A et al., 1990, J Immunol. 145: 3594-3600, but alsomannose and mannan are useful alternatives. Integral membrane proteinsfrom e.g. E. coli and other bacteria are also useful conjugationpartners. The traditional carrier molecules such as keyhole limpethemocyanin (KLH), tetanus toxoid, diphtheria toxoid, and bovine serumalbumin (BSA) are also preferred and useful conjugation partners.

Certain areas of native IL5 are believed to be superiorly suited forperforming modifications. It is predicted that modifications in at leastone of loops 1-3 or in the amino acid residues C-terminal to helix,D(said loops and said helix D corresponding to those shown in FIG. 3 forhuman and murine IL5) will be most likely to produce the desiredconstructs and vaccination results. Considerations underlying thesechosen areas are a) preservation of known and predicted B-cell epitopes,b) preservation of tertiary and quaternary structures etc, cf. also thediscussion in the preamble to the examples. At any rate, as discussedabove, it is fairly easy to screen a set of modified IL5 molecules whichhave all been subjected to introduction of a T-cell epitope in differentlocations.

Since the most preferred embodiments of the present invention involvesdown-regulation of human IL5, it is consequently preferred that the IL5polypeptide discussed above is a human IL5 polypeptide. In thisembodiment, it is especially preferred that the human IL5 polypeptidehas been modified by substituting at least one amino acid sequence inSEQ ID NO: 1 with at least one amino acid sequence of equal or differentlength and containing a foreign T_(H) epitope, wherein substituted aminoacid residues are selected from the group consisting of residues 87-90,residues 32-43, residues 59-64, residues 86-91, and residues 110-113.The rationale behind such constructs is discussed in detail in theexamples.

Formulation of IL5 and Modified IL5 Polypeptides

When effecting presentation of the IL5 polypeptide or the modified IL5polypeptide to an animal's immune system by means of administrationthereof to the animal, the formulation of the polypeptide follows theprinciples generally acknowledged in the art.

Preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like, and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines; cf. thedetailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously, intracutaneusly, intradermally,subdermally or intramuscularly. Additional formulations which aresuitable for other modes of administration include suppositories and, insome cases, oral, buccal, sublinqual, intraperitoneal, intravaginal,anal, epidural, spinal, and intracranial formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1-2%. Oral formulations include such normallyemployed excipients as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, and the like. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10-95% of active ingredient,preferably 25-70%. For oral formulations, cholera toxin is aninteresting formulation partner (and also a possible conjugationpartner).

The polypeptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include acid addition salts(formed with the free amino groups of the peptide) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to mount an immune response, and the degree of protectiondesired. Suitable dosage ranges are of the order of several hundredmicrograms active ingredient per vaccination with a preferred range fromabout 0.1 μg to 2,000 μg (even though higher amounts in the 1-10 mgrange are contemplated), such as in the range from about 0.5 μg to 1,000μg, preferably in the range from 1 μg to 500 μg and especially in therange from about 10 μg to 100 μg. Suitable regimens for initialadministration and booster shots are also variable but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These includeoral application on a solid physiologically acceptable base or in aphysiologically acceptable dispersion, parenterally, by injection or thelike. The dosage of the vaccine will depend on the route ofadministration and will vary according to the age of the person to bevaccinated and the formulation of the antigen.

Some of the polypeptides of the vaccine are sufficiently immunogenic ina vaccine, but for some of the others the immune response will beenhanced if the vaccine further comprises an adjuvant substance.

Various methods of achieving adjuvant effect for the vaccine are known.General principles and methods are detailed in “The Theory and PracticalApplication of Adjuvants”, 1995, Duncan E. S. Stewart-Tull (ed.), JohnWiley & Sons Ltd, ISBN 0-471-95170-6, and also in “Vaccines: NewGeneration Immunological Adjuvants”, 1995, Gregoriadis G et al. (eds.),Plenum Press, New York, ISBN 0-306-45283-9, both of which are herebyincorporated by reference herein.

It is especially preferred to use an adjuvant which can be demonstratedto facilitate breaking of the autotolerance to autoantigens; in fact,this is essential in cases where unmodified IL5 is used as the activeingredient in the autovaccine. Non-limiting examples of suitableadjuvants are selected from the group consisting of an immune targetingadjuvant; an immune modulating adjuvant such as a toxin, a cytokine, anda mycobacterial derivative; an oil formulation; a polymer; a micelleforming adjuvant; a saponin; an immunostimulating complex matrix (ISCOMmatrix); a particle; DDA; aluminium adjuvants; DNA adjuvants; γ-inulin;and an encapsulating adjuvant. In general it should be noted that thedisclosures above which relate to compounds and agents useful as first,second and third moieties in the analogues also refer mutatis mutandisto their use in the adjuvant of a vaccine of the invention.

The application of adjuvants include use of agents such as aluminiumhydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percentsolution in buffered saline, admixture with synthetic polymers of sugars(e.g. Carbopol®) used as 0.25 percent solution, aggregation of theprotein in the vaccine by heat treatment with temperatures rangingbetween 70 to 101° C. for 30 second to 2 minute periods respectively andalso aggregation by means of cross-linking agents are possible.Aggregation by reactivation with pepsin treated antibodies (Fabfragments) to albumin, mixture with bacterial cells such as C. parvum orendotoxins or lipopolysaccharide components of gram-negative bacteria,emulsion in physiologically acceptable oil vehicles such as mannidemono-oleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (Fluosol-DA) used as a block substitute may also beemployed. Admixture with oils such as squalene and IFA is alsopreferred.

According to the invention DDA (dimethyldioctadecylammonium bromide) isan interesting candidate for an adjuvant as is DNA and γ-inulin, butalso Freund's complete and incomplete adjuvants as well as quillajasaponins such as QuilA and QS21 are interesting as is RIBI. Furtherpossibilities are monophosphoryl lipid A (MPL), the above mentioned C3and C3d, and muramyl dipeptide (MDP).

Liposome formulations are also known to confer adjuvant effects, andtherefore liposome adjuvants are preferred according to the invention.

Also immunostimulating complex matrix type (ISCOMO matrix) adjuvants arepreferred choices according to the invention, especially since it hasbeen shown that this type of adjuvants are capable of up-regulating MHCClass II expression by APCs. An ISCOM® matrix consists of (optionallyfractionated) saponins (triterpenoids) from Quillaja saponaria,cholesterol, and phospholipid. When admixed with the immunogenicprotein, the resulting particulate formulation is what is known as anISCOM particle where the saponin constitutes 60-70% w/w, the cholesteroland phospholipid 10-15% w/w, and the protein 10-15% w/w. Detailsrelating to composition and use of immunostimulating complexes can e.g.be found in the above-mentioned text-books dealing with adjuvants, butalso Morein B et al., 1995, Clin. Immunother. 3: 461-475 as well as BarrIG and Mitchell GF, 1996, Immunol. and Cell Biol. 74: 8-25 (bothincorporated by reference herein) provide useful instructions for thepreparation of complete immunostimulating complexes.

Another highly interesting (and thus, preferred) possibility ofachieving adjuvant effect is to employ the technique described inGosselin et al., 1992 (which is hereby incorporated by referenceherein). In brief, the presentation of a relevant antigen such as anantigen of the present invention can be enhanced by conjugating theantigen to antibodies (or antigen binding antibody fragments) againstthe Fcγ receptors on monocytes/macrophages. Especially conjugatesbetween antigen and anti-FcγRI have been demonstrated to enhanceimmunogenicity for the purposes of vaccination.

Other possibilities involve the use of the targeting and immunemodulating substances (i.a. cytokines) mentioned above as candidates forthe first and second moieties in the modified versions of IL5. In thisconnection, also synthetic inducers of cytokines like poly I:C arepossibilities.

Suitable mycobacterial derivatives are selected from the groupconsisting of muramyl dipeptide, complete Freund's adjuvant, RIBI, and adiester of trehalose such as TDM and TDE.

Suitable immune targeting adjuvants are selected from the groupconsisting of CD40 ligand and CD40 antibodies or specifically bindingfragments thereof (cf. the discussion above), mannose, a Fab fragment,and CTLA-4.

Suitable polymer adjuvants are selected from the group consisting of acarbohydrate such as dextran, PEG, starch, mannan, and mannose; aplastic polymer such as; and latex such as latex beads.

Yet another interesting way of modulating an immune response is toinclude the IL5 immunogen (optionally together with adjuvants andpharmaceutically acceptable carriers and vehicles) in a “virtual lymphnode” (VLN) (a proprietary medical device developed by ImmunoTherapy,Inc., 360 Lexington Avenue, New York, N.Y. 10017-6501). The VLN (a thintubular device) mimics the structure and function of a lymph node.Insertion of a VLN under the skin creates a site of sterile inflammationwith an upsurge of cytokines and chemokines. T- and B-cells as well asAPCs rapidly respond to the danger signals, home to the inflamed siteand accumulate inside the porous matrix of the VLN. It has been shownthat the necessary antigen dose required to mount an immune response toan antigen is reduced when using the VLN and that immune protectionconferred by vaccination using a VLN surpassed conventional immunizationusing Ribi as an adjuvant. The technology is i.a. described briefly inGelber C et al., 1998, “Elicitation of Robust Cellular and HumoralImmune Responses to Small Amounts of Immunogens Using a Novel MedicalDevice Designated the Virtual Lymph Node”, in: “From the Laboratory tothe Clinic, Book of Abstracts, Oct. 12^(th)-15^(th) 1998, SeascapeResort, Aptos, Calif.”.

It is expected that the vaccine should be administered at least once ayear, such as at least 1, 2, 3, 4, 5, 6, and 12 times a year. Morespecifically, 1-12 times per year is expected, such as 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 times a year to an individual in need thereof. Ithas previously been shown that the memory immunity induced by the use ofthe preferred autovaccines according to the invention is not permanent,and therefor the immune system needs to be periodically challenged withthe analogues.

Due to genetic variation, different individuals may react with immuneresponses of varying strength to the same polypeptide. Therefore, thevaccine according to the invention may comprise several differentpolypeptides in order to increase the immune response, cf. also thediscussion above concerning the choice of foreign T-cell epitopeintroductions. The vaccine may comprise two or more polypeptides, whereall of the polypeptides are as defined above.

The vaccine may consequently comprise 3-20 different modified orunmodified polypeptides, such as 3-10 different polypeptides. However,normally the number of polypeptides will be sought kept to a minimumsuch as 1 or 2 polypeptides.

Nucleic Acid Vaccination

As an alternative to classic administration of a peptide-based vaccine,the technology of nucleic acid vaccination (also known as “nucleic acidimmunisation”, “genetic immunisation”, and “gene immunisation”) offers anumber of attractive features.

First, in contrast to the traditional vaccine approach, nucleic acidvaccination does not require resource consuming large-scale productionof the immunogenic agent (e.g. in the form of industrial scalefermentation of microorganisms producing modified IL5). Furthermore,there is no need to device purification and refolding schemes for theimmunogen. And finally, since nucleic acid vaccination relies on thebiochemical apparatus of the vaccinated individual in order to producethe expression product of the nucleic acid introduced, the optimumposttranslational processing of the expression product is expected tooccur; this is especially important in the case of autovaccination,since, as mentioned above, a significant fraction of the original IL5B-cell epitopes should be preserved in the modified molecule, and sinceB-cell epitopes in principle can be constituted by parts of any(bio)molecule (e.g. carbohydrate,: lipid, protein etc.). Therefore,native glycosylation and lipidation patterns of the immunogen may verywell be of importance for the overall immunogenicity and this isexpected to be ensured by having the host producing the immunogen.

Hence, a preferred embodiment of the invention comprises effectingpresentation of modified IL5 to the immune system by introducing nucleicacid(s) encoding the modified IL5 into the animal's cells and therebyobtaining in vivo expression by the cells of the nucleic acid(s)introduced.

In this embodiment, the introduced nucleic acid is preferably DNA whichcan be in the form of naked DNA, DNA formulated with charged oruncharged lipids, DNA formulated in liposomes, DNA included in a viralvector, DNA formulated with a transfection-facilitating protein orpolypeptide, DNA formulated with a targeting protein or polypeptide, DNAformulated with Calcium precipitating agents, DNA coupled to an inertcarrier molecule, DNA encapsulated in a polymer, e.g. in PLGA (cf. themicroencapsulation technology described in WO 98/31398) or in chitin orchitosan, and DNA formulated with an adjuvant. In this context it isnoted that practically all considerations pertaining to the use ofadjuvants in traditional vaccine formulation apply for the formulationof DNA vaccines. Hence, all disclosures herein which relate to use ofadjuvants in the context of polypeptide based vaccines apply mutatismutandis to their use in nucleic acid vaccination technology.

As for routes of administration and administration schemes ofpolypeptide based vaccines which have been detailed above, these arealso applicable for the nucleic acid vaccines of the invention and alldiscussions above pertaining to routes of administration andadministration schemes for polypeptides apply mutatis mutandis tonucleic acids. To this should be added that nucleic acid vaccines cansuitably be administered intraveneously and intraarterially.Furthermore, it is well-known in the art that nucleic acid vaccines canbe administered by use of a so-called gene gun, and hence also this andequivalent modes of administration are regarded as part of the presentinvention. Finally, also the use of a VLN in the administration ofnucleic acids has been reported to yield good results, and thereforethis particular mode of administration is particularly preferred.

Furthermore, the nucleic acid(s) used as an immunization agent cancontain regions encoding the 1^(st), 2^(nd) and/or 3^(rd) moieties, e.g.in the form of the immunomodulating substances described above such asthe cytokines discussed as useful adjuvants. A preferred version of thisembodiment encompasses having the coding region for the analogue and thecoding region for the immunomodulator in different reading frames or atleast under the control of different promoters. Thereby it is avoidedthat the analogue or epitope is produced as a fusion partner to theimmunomodulator. Alternatively, two distinct nucleotide fragments can beused, but this is less preferred because of the advantage of ensuredco-expression when having both coding regions included in the samemolecule.

Accordingly, the invention also relates to a composition for inducingproduction of antibodies against IL5, the composition comprising

a nucleic acid fragment or a vector of the invention (cf. the discussionof vectors below), and

a pharmaceutically and immunologically acceptable vehicle and/or carrierand/or adjuvant as discussed above.

Under normal circumstances, the IL5 variant-encoding nucleic acid isintroduced in the form of a vector wherein expression is under controlof a viral promoter. For more detailed discussions of vectors and DNAfragments according to the invention, cf. the discussion below. Also,detailed disclosures relating to the formulation and use of nucleic acidvaccines are available, cf. Donnelly J J et al, 1997, Annu. Rev.Immunol. 15: 617-648 and Donnelly J J et al., 1997, Life Sciences 60:163-172. Both of these references are incorporated by reference herein.

Live Vaccines

A third alternative for effectingipresentation of modified IL5 to theimmune system is the use of live vaccine technology. In livevaccination, presentation to the immune system is effected byadministering, to the animal, a non-pathogenic microorganism which hasbeen transformed with a nucleic acid fragment encoding a modified IL5 orwith a vector incorporating such a nucleic acid fragment. Thenon-pathogenic microorganism can be any suitable attenuated bacterialstrain (attenuated by means of passaging or by means of removal ofpathogenic expression products by recombinant DNA technology), e.g.Mycobacterium bovis BCG., non-pathogenic Streptococcus spp., E. coli,Salmonella spp., Vibrio cholerae, Shigella, etc. Reviews dealing withpreparation of state-of-the-art live vaccines can e.g. be found inSaliou P, 1995, Rev. Prat. 45: 1492-1496 and Walker P D, 1992, Vaccine10: 977-990, both incorporated by reference herein. For details aboutthe nucleic acid fragments and vectors used in such live vaccines, cf.the discussion below.

As an alternative to bacterial live vaccines, the nucleic acid fragmentof the invention discussed below can be incorporated in a non-virulentviral vaccine vector such as a vaccinia strain or any other suitable poxvirus.

Normally, the non-pathogenic microorganism or virus is administered onlyonce to the animal, but in certain cases it may be necessary toadminister the microorganism more than once in a lifetime in order tomaintain protective immunity. It is even contemplated that immunizationschemes as those detailed above for polypeptide vaccination will beuseful when using live or virus vaccines.

Alternatively, live or virus vaccination is combined with previous orsubsequent polypeptide and/or nucleic acid vaccination. For instance, itis possible to effect primary immunization with a live or virus vaccinefollowed by subsequent booster immunizations using the polypeptide ornucleic acid approach.

The microorganism or virus can be transformed with nucleic acid(s)containing regions encoding the 1^(st), 2^(nd) and/or 3^(rd) moieties,e.g. in the form of the immunomodulating substances described above suchas the cytokines discussed as useful adjuvants. A preferred version ofthis embodiment encompasses having the coding region for the analogueand the coding region for the immunomodulator in different readingframes or at least under the control of different promoters. Thereby itis avoided that the analogue or epitopes are produced as fusion partnersto the immunomodulator. Alternatively, two distinct nucleotide fragmentscan be used as transforming agents. Of course, having the 1^(st) and/or2^(nd) and/or 3^(rd) moieties in the same reading frame can provide asan expression product, an analogue of the invention, and such anembodiment is especially preferred according to the present invention.

Use of the Method of the Invention in Disease Treatment

As will be appreciated from the discussions above, the provision of themethod of the invention allows for control of diseases characterized byeosinophilia. In this context, asthma is the key target for theinventive method but also other chronic allergic conditions such asmultiple allergy and allergic rhinitis are feasible targets fortreatment/amelioration. Hence, an important embodiment of the method ofthe invention for down-regulating IL5 activity comprises treating and/orpreventing and/or ameliorating asthma or other chronic allergicconditions characterized by eosinophilia, the method comprisingdown-regulating IL5 activity according to the method of the invention tosuch an extent that the number of eosinophil cells is significantlyreduced.

In the present context such a significant reduction in eosinophil cellnumbers is at least 20% compared to the eosinophil number prior totreatment, but higher percentages are contemplated, such as at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% and even at least 90%. The reduction may be systemic or, more often,locally in e.g. the lungs.

Eosinophil cell numbers are determined by methods known in the art,typically using microscopy of a suitable sample (such as a BAL fluid)and counting the number of eosinophil cells manually under microscope.Alternatively, eosinophil numbers can be counted using flow cytometricmethods or any other convenient method of cytometry capable ofdistinguishing eosinophils.

Peptides, Polypeptides, and Compositions of the Invention

As will be apparent from the above, the present invention is based onthe concept of immunising individuals against the IL5 antigen in orderto indirectly obtain a reduction in eosinophil cell numbers. Thepreferred way of obtaining such an immunization is to use modifiedversions of IL5, thereby providing molecules which have not previouslybeen disclosed in the art.

It is believed that the modified IL5 molecules discussed herein areinventive in their own right, and therefore an important part of theinvention pertains to an IL5 analogue which is derived from an animalIL5 wherein is introduced a modification which has as a result thatimmunization of the animal with the analogue induces production ofantibodies cross-reacting with the unmodified IL5 polypeptide.Preferably, the nature of the modification conforms with the types ofmodifications described above when discussing various embodiments of themethod of the invention when using modified IL5. Hence, any disclosurepresented herein pertaining to modified IL5 molecules are relevant forthe purpose of describing the IL5 analogues of the invention, and anysuch disclosures apply mutatis mutandis to the description of theseanalogues.

It should be noted that preferred modified IL5 molecules comprisemodifications which results in a polypeptide having a sequence identityof at least 70% with IL5 or with a subsequence thereof of at least 10amino acids in length. Higher sequence identities are preferred, e.g. atleast 75% or even at least 80% or 85%. The sequence identity forproteins and nucleic acids can be calculated as(N_(ref)−N_(dif))·100/N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence identity of 75% with thesequence AATCAATC (N_(dif)=2 and N_(ref)=8).

The invention also pertains to compositions useful in exercising themethod of the invention. Hence, the invention also relates to animmunogenic composition comprising an immunogenically effective amountof an IL5 polypeptide which is a self-protein in an animal, said IL5polypeptide being formulated together with an immunologically acceptableadjuvant so as to break the animal's autotolerance towards the IL5polypeptide, the composition further comprising a pharmaceutically andimmunologically acceptable vehicle and/or carrier. In other words, thispart of the invention pertains to the formulations of naturallyoccurring IL5 polypeptides which have been described in connection withembodiments of the method of the invention.

The invention also relates to an immunogenic composition comprising animmunologically effective amount of an IL5 analogue defined above, saidcomposition further comprising a pharmaceutically and immunologicallyacceptable diluent and/or vehicle and/or carrier and/or excipient andoptionally an adjuvant. In other words, this part of the inventionconcerns formulations of modified IL5, essentially as describedhereinabove. The choice of adjuvants, carriers, and vehicles isaccordingly in line with what has been discussed above when referring toformulation of modified and unmodified IL5 for use in the inventivemethod for the down-regulation of IL5.

The polypeptides are prepared according to methods well-known in theart. Longer polypeptides are normally prepared by means of recombinantgene technology including introduction of a nucleic acid sequenceencoding the IL5 analogue into a suitable vector, transformation of asuitable host cell with the vector, expression of the nucleic acidsequence, recovery of the expression product from the host cells ortheir culture supernatant, and subsequent purification and optionalfurther modification, e.g. refolding or derivatization.

Shorter peptides are preferably prepared by means of the wellknowntechniques of solid- or liquid-phase peptide synthesis. However, recentadvances in this technology has rendered possible the production offull-length polypeptides and proteins by these means, and therefore itis also within the scope of the present invention to prepare the longconstructs by synthetic means.

Nucleic Acid Fragments and Vectors of the Invention

It will be appreciated from the above disclosure that modified IL5polypeptides can be prepared by means of recombinant gene technology butalso by means of chemical synthesis or semisynthesis; the latter twooptions are especially relevant when the modification consists incoupling to protein carriers (such as KLH, diphtheria toxoid, tetanustoxoid, and BSA) and non-proteinaceous molecules such as carbohydratepolymers and of course also when the modification comprises addition ofside chains or side groups to an IL5 polypeptide-derived peptide chain.

For the purpose of recombinant gene technology, and of course also forthe purpose of nucleic acid immunization, nucleic acid fragmentsencoding modified IL5 are important chemical products. Hence, animportant part of the invention pertains to a nucleic acid fragmentwhich encodes an IL5 analogue, i.e. an IL5 derived polypeptide whicheither comprises the natural IL5 sequence to which has been added orinserted a fusion partner or, preferably an IL5 derived polypeptidewherein has been introduced a foreign T-cell epitope by means ofinsertion and/or addition, preferably by means of substitution and/ordeletion. The nucleic acid fragments of the invention are either DNA orRNA fragments.

The nucleic acid fragments of the invention will normally be inserted insuitable vectors to form cloning or expression vectors carrying thenucleic acid fragments of the invention; such novel vectors are alsopart of the invention. Details concerning the construction of thesevectors of the invention will be discussed in context of transformedcells and microorganisms below. The vectors can, depending on purposeand type of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors of the invention are capable of autonomousreplication, thereby enabling high copy-numbers for the purposes ofhigh-level expression or high-level replication for subsequent cloning.

The general outline of a vector of the invention comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma) of or integration into the membrane of the polypeptidefragment, the nucleic acid fragment of the invention, and optionally anucleic acid sequence encoding a terminator. When operating withexpression vectors in producer strains or cell-lines it is for thepurposes of genetic stability of the transformed cell preferred that thevector when introduced into a host cell is integrated in the host cellgenome. In contrast, when working with vectors to be used for effectingin vivo expression in an animal (i.e. when using the vector in DNAvaccination) it is for security reasons preferred that the vector is notincapable of being integrated in the host cell genome; typically, nakedDNA or non-integrating viral vectors are used, the choices of which arewell-known to the person skilled in the art.

The vectors of the invention are used to transform host cells to producethe modified IL5 polypeptide of the invention. Such transformed cells,which are also part of the invention, can be cultured cells or celllines used for propagation of the nucleic acid fragments and vectors ofthe invention, or used for recombinant production of the modified IL5polypeptides of the invention. Alternatively, the transformed cells canbe suitable live vaccine strains wherein the nucleic acid fragment (onesingle or multiple copies) have been inserted so as to effect secretionor integration into the bacterial membrane or cell-wall of the modifiedIL5.

Preferred transformed cells of the invention are microorganisms such asbacteria (such as the species Escherichia [e.g. E. coli], Bacillus [e.g.Bacillus subtilis], Salmonella, or Mycobacterium [preferablynon-pathogenic, e.g. M. bovis BCG]), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells arederived from a multi-cellular organism such as a fungus, an insect cell,a plant cell, or a mammalian cell. Most preferred are cells derived froma human being, cf. the discussion of cell lines and vectors below.Recent results have shown great promise in the use of a commerciallyavailable Drosophila melanogaster cell line (the Schneider 2 (S₂)cellline and vector system available from Invitrogen) for the recombinantproduction of IL5 analogues of the invention, and therefore thisexpression system is particularly preferred.

For the purposes of cloning and/or optimized expression it is preferredthat the transformed cell is capable of replicating the nucleic acidfragment of the invention. Cells expressing the nucleic fragment arepreferred useful embodiments of the invention; they can be used forsmall-scale or large-scale preparation of the modified IL5 or, in thecase of non-pathogenic bacteria, as vaccine constituents in a livevaccine.

When producing the modified IL5 of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is either exported out into 1the culture medium orcarried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragmentencoding the modified IL5. Preferably, this stable cell line secretes orcarries the IL5 analogue of the invention, thereby facilitatingpurification thereof.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with the hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies (see, e.g., Bolivar et al., 1977). The pBR322 plasmid containsgenes for ampicillin and tetracycline resistance and thus provides easymeans for identifying transformed cells. The pBR plasmid, or othermicrobial plasmid or phage must also contain, or be modified to contain,promoters which can be used by the prokaryotic microorganism forexpression.

Those promoters most commonly used in prokaryotic recombinant DNAconstruction include the B-lactamase (penicillinase) and lactosepromoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel etal., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979;EP-A-0 036 776). While these are the most commonly used, other microbialpromoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors (Siebwenlist etal., 1980). Certain genes from prokaryotes may be expressed efficientlyin E. coli from their own promoter sequences, precluding the need foraddition of another promoter by artificial means.

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and here the promoter should be capable of drivingexpression. Saccharomyces cerevisiase, or common baker's yeast is themost commonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb et al.,1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmidalready contains the trpl gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpllesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or otherglycolytic-enzymes (Hess et al., 1968; Holland et al., 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda(SF) cells (commercially available as complete expression systems fromi.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,U.S.A. and from Invitrogen), and MDCK cell lines. In the presentinvention, an especially preferred cell line is S2 available fromInvitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication (Fierset al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g., Polyoma, Adeno, VSV, BPV) or may be provided bythe host cell chromosomal replication mechanism. If the vector isintegrated into the host cell chromosome, the latter is oftensufficient.

Identification of Useful IL5 Analogues

It will be clear to the skilled person that not all variants ormodifications of native IL5 will have the ability to elicit antibodiesin an animal which are cross-reactive with the native form. It is,however, not difficult to set up an effective standard screen formodified IL5 molecules which fulfill the minimum requirements forimmunological reactivity discussed herein. Hence, another part of theinvention concerns a method for the identification of a modified IL5polypeptide which is capable of inducing antibodies against unmodifiedIL5 in an animal species where the unmodified IL5 polypeptide is aself-protein, the method comprising

preparing, by means of peptide synthesis or by molecular biologicalmeans, a set of mutually distinct modified IL5 polypeptides whereinamino acids have been added to, inserted in, deleted from, orsubstituted into the amino acid sequence of an IL5 polypeptide of theanimal species thereby giving rise to amino acid sequences in the setwhich comprise T-cell epitopes which are and foreign to the animalspecies, or preparing a set of nucleic acid fragments encoding the setof mutually distinct modified IL5 polypeptides,

testing members of the set for their ability to induce production ofantibodies by the animal species against the unmodified IL5, and

identifying and optionally isolating the member(s) of the set whichsignificantly induces antibody production against unmodified IL5 in theanimal species, or identifying and optionally isolating the polypeptideexpression products encoded by members of the set of nucleic acidfragments which significantly induces antibody production againstunmodified IL5 polypeptide in the animal species.

In this context, the “set of mutually distinct modified IL5polypeptides” is a collection of non-identical modified IL5 polypeptideswhich have e.g. been selected on the basis of the criteria discussedabove (e.g. in combination with studies of circular dichroism, NMRspectra, and/or X-ray diffraction patterns). The set may consist of onlya few members but it is contemplated that the set may contain severalhundred members. Likewise, the set of nucleic acid fragments is acollection of non-identical nucleic acid fragments, each encoding amodified IL5 polypeptide selected in the same manner.

The test of members of the set can ultimately be performed in vivo, buta number of in vitro tests can be applied which narrow down the numberof modified molecules which will serve the purpose of the invention.

Since the goal of introducing the foreign T-cell epitopes is to supportthe B-cell response by T-cell help, a prerequisite is that T-cellproliferation is induced by the modified IL5. T-cell proliferation canbe tested by standardized proliferation assays in vitro. In short, asample enriched for T-cells is obtained from a subject and subsequentlykept in culture. The cultured T-cells are contacted with APCs of thesubject which have previously taken up the modified molecule andprocessed it to present its T-cell epitopes. The proliferation ofT-cells is monitored and compared to a suitable control (e.g. T-cells inculture contacted with APCs which have processed intact, native IL5).Alternatively, proliferation can be measured by determining theconcentration of relevant cytokines released by the T-cells in responseto their recognition of foreign T-cells.

Having rendered highly probable that at least one modified IL5 of theset is capable of inducing antibody production against IL5, it ispossible to prepare an immunogenic composition comprising at least onemodified IL5 polypeptide which is capable of inducing antibodies againstunmodified IL5 in an animal species where the unmodified IL5 polypeptideis a self-protein, the method comprising admixing the member(s) of theset which significantly induces production of antibodies in the animalspecies which are reactive with IL5 with a pharmaceutically andimmunologically acceptable carrier and/or vehicle and/or diluent and/orexcipient, optionally in combination with at least one pharmaceuticallyand immunologically acceptable adjuvant.

Likewise, it is also possible to prepare an immunogenic compositionwhich as an immunogen contains a nucleic acid fragment encoding aimmunogenic IL5 analogue, cf. the discussion of nucleic acid vaccinationabove.

The above aspects of the invention are conveniently carried out byinitially preparing a number of mutually distinct nucleic acid sequencesor vectors of the invention, inserting these into appropriate expressionvectors, transforming suitable host cells with the vectors, andexpressing the nucleic acid sequences of the invention. These steps canbe followed by isolation of the expression products. It is preferredthat the nucleic acid sequences and/or vectors are prepared by methodscomprising exercise of a molecular amplification technique such as PCRor by means of nucleic acid synthesis.

PREAMBLE TO EXAMPLES Vaccine Design

The exemplary candidates for an IL5 autovaccine are constructedaccording to the AutoVac™ concept (described in detail in WO 95/05849)by substitution with known promiscuous T cell epitopes into the humanIL5 wild type protein. The substitutions are peptide substitutions,where the inserted peptide may be of the same or different length thanthe deleted peptide in the wild-type sequence.

For initial proof of concept by in vivo testing and screening, it wasdecided to prepare the constructs in the murine IL5 sequence. By way ofexample, the tetanus toxoid epitopes P2 (SEQ ID NO: 23) and P30 (SEQ IDNO: 24) are used as substituting peptides, but any other suitablepeptide containing or constituting a promiscuous T_(H) epitope could,according to the present invention, be used.

It should be emphasized that the size of the molecule (115 res.)compared to the size of the substitutions (15 or 21 residues for P2 andP30, respectively) strongly limits the possible sites of structuralnon-destructive inserts. As the disulfide bridges are important, but notimperative, for the dimerization, some variants are made in pairs +/−elimination of the cysteines.

In the construction of the candidate molecules, two basic parametershave been considered. First, it is attempted to conserve a maximumfraction of the three-dimensional structure of the wild type hIL5,thereby conserving the native B-cell epitope repertoire. This issupported by Dickason et al., (1994) who demonstrated that IL5 B-cellepitopes known to be neutralising are conformational. Conservation ofthe tertiary structure is sought achieved by introducing themodifications at structurally “neutral” sites, such as loops or separatesegments. The fact that the N-terminal helix “A” together with thehelices “B” and “C” are able to fold into a quaternary structure with asecond molecule, indicates that these 3 helices constitute a stablefolding-scaffold.

Second, the biological activity in relation to the vaccine concept hasbeen considered. In general, an inactive construct is preferable with aview to reducing putative toxic effects of the molecules and in generalfor evaluating the immune response. On the other hand, the optimumneutralising antibodies should theoretically exhibit specificity for thepart of IL5 that interacts with the IL5R. This is most likely achievedby immunising with an active variant. Finally, it is not impossible thatthe biological effect of IL5 on the immune system might act as anenhancer on the immune response, thus improving the overall effect.Based on Applicant's previous experiences with other molecules, however,the majority of “theoretically possible active” constructs is expectedto have low or no activity.

Therefore, all variants suggested are potentially active but can, ifdesirable, with relative ease be rendered inactive by hindering theformation of the active dimer or by alterations in the areas of the “A”-and “D”-helices that are involved in the receptor binding/activation.

In summary, the above considerations of structure conservation andbiological activity defines the target areas as any one of loops 1-3 aswell as the C-terminal flexible area.

Loop 3 is selected as the primary target area since it is structurallyseparated from the assumed tri-helical folding scaffold. As it isfurthermore possible to produce a biologically active monomer, byelongation of loop 3 (Dickason, 1996), this area holds the possibilitiesfor generating all types of variants: monomer/dimer andactive/inactivated.

“Loop 1” is a second area containing a non-helical stretch of a suitablelength for substitutions. Variants from this region would theoreticallybe active only if capable of dimerising, but since the length of thewild-type loop makes it rather flexible it is reasonable to expect acorrect folding of the protein after substitution.

Variants containing substitutions in the “loop 2” area will also only beactive as dimers. The area that can be substituted is short compared tothe inserts and has a central position in the assumed folding scaffold,two characteristics of loop 2 which might be of hindrance to the correctfolding of the protein after substitution. On the other hand, loop 2 issituated opposite to the area interacting with the IL5R, resulting in anexpected optimum presentation of the wild-type neutralising epitopes ifthe modified protein is correctly folded.

Finally, inserts in the C-terminal flexible region following “helix D”are proposed. From a protein structure point of view this conceptappears fairly safe, but it is likely that modifications in this regionwill affect both dimerization and biological activity (if the modifiedprotein is dimerized) since the C-terminal is located in the area ofboth receptor binding and in the dimer interface.

The amino acid sequence of 10 variants initially constructed accordingto the above considerations are set forth as SEQ ID NOs: 2-11 and 13-22.Further variants constructed at a later stage are set forth in SEQ IDNOs: 27-59 (including both DNA nucleic acid sequences and amino acidsequences).

It should be noted, that all inserts except from the ones according toExample 2 are prepared so as to include flanking amino acid residuesthat are conserved from hIL5 to mIL5 in order to promote the process ofsuccessful transfer of positive constructs from mice to man.

In the following examples, positions for substitution are indexedaccording to the murine amino acid residue sequence numbers; thecorresponding human positions are given in parentheses.

Example 1

Variants With P2 Substituting Positions in Loop 3 While PreservingCys84(86)

The P2 epitope (SEQ ID NO: 23) is substituted into loop 3 while avoidingelimination of Cys84(86). These variants (SEQ ID NOs: 2 and 28 (human),where amino acids 87-90 or 88-91 are substituted and 13 and 46 (murine)where amino acids 85-88 pr 86-89 are substituted) are potentially activeas both monomers (due to the elongation of loop 3) and as dimers. SEQ IDNos: 28 and 46 are also denoted hIL5.1 and mIL5.1, respectively.

Example 2

Variants With P2 Substituting Positions in Loop 1 While PreservingCys42(44)

The P2 epitope (SEQ ID NO: 23) is substituted into loop 1 while avoidingelimination of Cys42(44). These variants (SEQ ID NOs: 3 and 36 (human)where amino acids 32-43 or 33-43 are substituted and 14 and 56 (murine)where amino acids 30-41 or 31-41 are substituted) are potentially activeas dimers only. SEQ ID Nos: 36 and 56 are also denoted hIL5.5 andmIL5.5, respectively.

Example 3

Variants with P2 Substituting Positions in Loop 2

The P2 epitope (SEQ ID NO: 23) is substituted into loop 2. Thesevariants (SEQ ID NOs: 4 and 34 (human) where amino acids 59-64 aresubstituted and 15 and 50 (murine) where amino acids 57-62 aresubsituted) are potentially active as dimers only. SEQ ID Nos: 34 and 50are also denoted hIL5.4 and mIL5.4, respectively.

Example 4

Variants With P2 Substituting Positions in Loop 3 While EliminatingCys84(86)

The P2 epitope (SEQ ID NO: 23) is substituted into loop 3 whileeliminating Cys84(86). These variants (SEQ ID NOs: 5 and 38 (human)where amino acids 86-91 are substituted and 16 and 54 (murine) whereamino acids 84-89 are substituted) are in principle similar to thevariants of type #1 (SEQ ID NOs: 2 and 28 and 13 and 46), but thegeneration of monomer products has been facilitated by inhibiting theformation of disulfide bridging and adjusting the length of loop 3. SEQID Nos: 38 and 54 are also denoted hIL5.6 and mIL5.6, respectively.

Example 5

Variants With P2 Substituting Positions 108-111(110-113) in theC-terminus

The P2 epitope (SEQ ID NO: 23) is substituted into the C-terminal areasucceeding helix D. These variants (SEQ ID NOs: 6 and 17) arepotentially active as a dimer only.

Example 6

Variants With P30 Substituting Positions in Loop 3 While PreservingCys84 (86)

The P30 epitope (SEQ ID NO: 24) is substituted into loop 3 avoidingelimination of Cys84(86). These variants (SEQ ID NOs: 7 and 40 (human)where amino acids 88-91 or 87-90 are substituted and 18 and 58 (murine)where amino acids 85-88 or 86-89 are substituted) are potentially activeboth as monomers (due to the elongation of loop 3) and as dimers. SEQ IDNos: 40 and 58 are also denoted hIL5.7 and mIL5.7, respectively.

Example 7

Variants With P30 Substituting Positions in Loop 1 While PreservingCys42(44)

The P30 epitope (SEQ ID NO: 24) is substituted into loop 1, avoidingelimination of Cys42(44). These variants (SEQ ID NOs: 8 and 30 (human)where amino acids 32-43 are substituted and 19 and 48 (murine) whereamino acids 30-41 are substituted) are potentially active as dimersonly. SEQ ID Nos: 30 and 48 are also denoted hIL5.2 and mIL5.2,respectively.

Example 8

Variants With P30 Substituting Positions in Loop 2

The P30 epitope (SEQ ID NO: 24) is substituted into loop 2. Thesevariants (SEQ ID NOs: 9 and 20 where amino acids 59-64 and 57-62 aresubstituted, respectively) are potentially active as dimers only.

Example 9

Variants With P30 Substituting Positions in the C Terminus

The P30 epitope (SEQ ID NO: 24) is substituted into the C-terminal areasucceeding helix D. These variants (SEQ ID NOs: 10 and 21 where aminoacids 110-113 and 108-111 are substituted, respectively) are potentiallyactive as dimers only.

Example 10

Variants With P2 Substituting Positions 84-89 (86-91) and P30Substituting Positions 110-113

The P2 epitope (SEQ ID NO: 23) is substituted into loop 3 eliminatingCys84(86) and the P30 epitope (SEQ ID NO: 24) is substituted into theC-terminal area succeeding helix-D. These variants (SEQ ID NOs: 11 and22) are together with variants of type #12 the only ones containing bothepitopes and are potentially active monomers.

Example 11

Variants With P30 Substituting Positions in Loop 3 While EliminatingCys84 (86)

The P2 epitope (SEQ ID NO: 24) is substituted into loop 3 whileeliminating Cys84(86). These variants (SEQ ID NOs: 42 (human) whereamino acids 86-91 are substituted and 58 (murine) where amino acids84-89 are substituted) are in principle similar to the variants of type#6, but the generation of monomer products has been facilitated byinhibiting the formation of disulfide bridging and adjusting the lengthof loop 3. SEQ ID Nos: 42 and 58 are also denoted hIL5.12 and mIL5.12,respectively.

Example 12

Variants With P2 and P30 Substituting Positions in Loop 3

The P2 (SEQ ID NO: 23) and P30 (SEQ ID NO: 24) epitopes are substitutedinto loop 3 while preserving Cys84(86). These variants (SEQ ID NOs: 44and 60 where amino acids 88-91 and 86-89 are substituted, respectively)contain both epitopes and are potentially active monomers. SEQ ID NOs:44 and 60 are also denoted hIL5.13 and mIL5.13, respectively.

Example 13

Choise of Expression System

Recombinant IL5 has been expressed in a number of different expressionsystems including yeast, insect cells and CHO cells (Tavernier et al.,1989).

According to the present invention, one suitable expression system is E.coli, based on previous studies reporting the use of this host forproduction hIL5 (Proudfoot et al., 1990, Graber et al., 1993). Therecombinant protein is expressed as inclusion bodies that are convertedinto the biologically active dimer upon purification and re-folding(e.g. using the generally applicable refolding methods disclosed in U.S.Pat. No. 5,739,281). The speed and simplicity of E. coli expressionallows immediate initiation of the production of protein when thegenetic constructs are ready, thus facilitating rapid generation ofmaterial to establish an in vivo proof of the IL5 autovaccine concept.

If for some reason the feasibility is found to be to low (e.g. low yieldfollowing re-folding, instability of the products or improvedpharmacokinetical parameters related to glycosylation etc), productionin yeast could be considered in a further development of theautovaccine.

Recently, promising results have: been obtained using the Drosophilamelanogaster expression system using S₂ cells (available fromInvitrogen) and at present this system is the preferred embodiment forexpression of the IL5 analogues of the invention.

IL5 variant protein was produced from S2 drosophila cells stablyexpressing the IL5 constructs. Several different transfection methodswere tested, and both Ca₂PO₄ and Lipofectin were chosen. Two differentsubclones of S2 cells were used and transfected with Ca₂PO₄ andLipofectin, respectively. The two clones were obtained from ATCC andLars Sondergaard of the University of Copenhagen, respectively. Usingboth methods suitable stable lines were selected expressing mIL5 andmIL5.1 proteins in the 2-10 mg/L range.

Materials & Methods:

S2 cells were grown and maintained in Schneider's medium (Sigma)containing 5-10% fetal calf serum (FCS), 0.1% pluronic F68 (Sigma),penicillin/streptomycin (Life Technologies) grown in shake flasks at 25°C. and 120 rpm.

Lipofectin transfections were performed in 250 ml or 1 l shake flasks.S2 cells were split to 2.5-3×10⁶/ml into 50 ml Excell 420 (JRHBiosciences) without antibiotics, and grown overnight in a 250 ml shakeflask. The next morning the Lipofectin reagents were prepared: tube 1)300-1200 μg plasmid DNA containing the gene of interest, plus 15-60 μgpCoHYGRO hygromycin selection plasmid (20:1 ratio of plasmids) in 15-45ml serum and supplement-free medium; tube 2) 1 ml Lipofectin in 5 mlserum and supplement-free medium. After 1 hour at room temperature,tubes 1 and 2 were mixed and rested for 15 minutes at room temperaturebefore gently adding to S2 cells. After growing cells overnight newmedia was added containing full suplements plus 150-300 pg/mlHygromycin.

Transient and stable lines were induced with either 500 μM coppersulfate or 10 μM cadmium chloride for 48-72 hours in serum-free Ex-cell420 medium (JRH Biosciences).

Results:

33 stable lines were generated by Ca₂PO₄ and 23 by Lipofectin. Theexpression yields varied from non-detectable up to 11 mg/L. Thefollowing table summarizes a few of the lines used for proteinproduction.

Expression result summary from best mIL5 S2 cell transfections.

Transfection Plasmid Construct S2 cells Method Yield p612IL5/His15/mIL5wt ATCC Ca₂PO₄ 3.5 mg/L p767 Bip/His15/mIL5wt LSLipofectin  11 mg/L p613 IL5/His15/mIL5.1 ATCC Ca₂PO₄ 2.6 mg/L p768Bip/His15/mIL5.1 ATCC Ca₂PO₄ 0* p614 ILS/His15/mIL5.5 LS Lipofectin 0**Expression plasmid contained sequence mutations.

Hence, S2 cells can be transfected by either calcium phosphateprecipitation or Lipofectin. Due to the difference in expression levelbetween plasmids p612 and p767, it seems that the Bip signal peptide isa more efficient leader sequence than the endogenous mIL5 leader in S2cells.

Example 14

Screening and Selection of the Modified Molecules

Following expression, the recombinant protein is purified andcharacterised. The characterisation of the autovaccine candidates willinclude analytical chromatography, iso-electric focussing (IEF),SDS-PAGE, amino acid composition analysis, N-terminal sequence analysis,mass spectrometry, low angle laser light scattering, standardspectroscopy, and Circular Dichroism to an extent that preciselydocument the relevant parameters defining the intended protein product.

The His tagged proteins have been purified using a two-step procedureuntil recently. However, the yield and purity were not as high asexpected after the final chelate-step. A new one-step purificationprocedure has been implied with 3 major advantages achieved: higheryield, higher through-put and higher purity of the final product.Cleavage conditions for removal of the histag have also beenestablished.

The Two-step IL5 Purification Procedure:

Expression of the protein is induced by addition of metal ions to themedia. These metal-ions have to be removed before application of theprotein to the chelate column. Thus, a total of 20 mM EDTA is added tocomplex the metal-ions and the supernatant is then passed over aSP-sepharose column to capture the protein. After washing to removeunbound protein, bound protein is eluted by a step-gradient of NaCl.This step serves two purposes: a concentrating step reducing the volumeby a factor of 30, and buffer-exchange.

Relevant fractions (as determined by SDS-PAGE) are pooled and furtherpurified on the metal chelate column.

The protein is applied to a Ni²⁺-charged chelate column and unboundprotein washed off. Bound protein is then eluted using an Imidazolegradient. All fractions, flow-through and EDTA-washes of the column, arethen checked by both SDS-PAGE and dot-blot.

Relevant fractions (as determined by SDS-PAGE and dot-blot) are pooledand dialyzed twice against 10×volume of PBS, pH adjusted to 6.9.

After filtration, the dialyzed material is concentrated until a suitableconcentration is achieved (preferably 1 mg/ml). Finally, the protein isaliqouted and stored at −20° C.

The following specific protocol has been applied:

1) The received supernatant is centrifuged at 2500 x g for 15 min (ifinfection has occurred, it needs centrifugation at 22000 x g for 30 min.The supernatant is then filtered using a 0.45 μm filter followed by a0.22 μm filter (sometimes it is necessary to filter through a 5 μmfilter first) The supernatant is then mixed 1:1 with buffer A (see step2) containing 40 mM EDTA, resulting in a final buffer composition of 0.2M NaH₂PO₄, 10% glycerol, 20 mM EDTA, pH 6.0 2) The filtered supernatantis subsequently applied to a SP-Sepha- rose column equilibrated inbuffer A. A total of 1-2 L (depend- ing on protein concentration, theabove holds for 1-10 mg IL5/ L) can be applied to an 80 ml column. Flowduring application: 1-2 ml/min (usually over night), the flow-through iscollected and saved for later analysis. Following application, thecolumn is washed with 2-3 column volumes (CV) of A-buffer until a stablebaseline is achieved. Bound protein is eluted using a step gradient:0-100-500-1000 mM NaCl, fractions of 10 ml are col- lected, flow is 10ml/min. Purification is performed at 5° C. The column is cleaned with 2CV 1 M NaOH, flow 5 ml/min after each run and re-equilibrated in bufferA. Buffer A: 0.2 M NaH₂PO₄, 10% glycerol, pH 6.0 Buffer B: 0.2 MNaH₂PO₄, 1 M NaCl, 40 mM Imidazole, 10% glycerol, pH 6.0 The sameprocedure is used for both wt and variants. All fractions, startingmaterial and flow-through are tested in dot-blot and SDS-PAGE. Thefractions containing IL5 are pooled and further purified using achelate-column.

The One-step IL5 Purification Procedure:

The supernatant is applied directly to a 70-ml chelate-column chargedwith ZnCl₂. After removal of the unbound material by washing, boundprotein (IL5 and contaminants) is eluted by applying a gradient ofImidazole. This method takes full advantage of the His tag giving aone-step purification procedure with a high degree of purity of thefinal product (>95%). Relevant fractions (as determined by SDS-PAGE anddot-blot) are pooled and dialyzed twice against 10×volume of PBS, pHadjusted to 6.9 and concentration of NaCl adjusted to 400 mM.

After filtration, the dialyzed material is concentrated until a suitableconcentration is achieved (preferably 1 mg/ml). Finally, the protein isaliqouted and stored at −20° C.

A specific protocol follows the following steps

1) The supernatant is filtered through a 0.45 μm filter to removeimpurities and diluted 1:1 with buffer A. A 70-ml Fast Flow chelatecolumn is rinsed with 5 CV water and then charged with 10 CV 10 mMZnCl₂, pH 7. After equilibration with 5 CV A-buffer, the sample isapplied using the pump (flow 10 ml/min). The flow-through is collectedand saved for later analysis. Bound protein is eluted using anImidazole-gradient going from 0 to 250 mM Imidazole over 30 CV. Finally,the column is stripped by 5 CV of buffer C. Fractions of 10 ml arecollected. Buffers: A: 20 mM NaH₂PO₄, 0.5 M NaCl, 10% glycerol, pH 7. B:20 mM NaH₂PO₄, 0.5 M NaCl, 10% glycerol, pH 7, 0.25 M imidazole C: 20 mMNaH₂PO₄, 0.5 M NaCl, 0.1 M EDTA pH 7.0. All fractions, flow-through andstarting material is tested in SDS-PAGE. 2) The purest fractions (asdetermined by SDS-PAGE) containing ILS are pooled (50 μl are saved forlater analysis) and dialyzed twice against 10 X volume of PBS, pHadjusted to 6.9. at 6° C., MWCO 12-14 kDa. The dialysate is filteredthrough a 0.22 μm fil- ter (50 μl is saved for later analysis) and A₂₈₀is measured using dialysis-buffer (filtered through 0.45 μm) asreference. The volume before and after dialysis is measured and samplesshowing the dialysis/concentrating step are saved for later analysis bySDS-PAGE (after step 3) 3) NaCl is added to the dialyzed protein until atotal concentra- tion of 400 mM and it is then concentrated using eitheran Ami- con apparatus (for volumes larger than 50 ml) or Vivaspin con-centrating device (for 10-50 ml). In both cases, the membrane issaturated with 10 ml PBS-buffer buffer before the sample is ap- plied.The sample should be concentrated until a concentration of preferably 1mg/ml is achieved (as measured by A₂₈₀). The dialyzed, concentratedsample is filtered through a 0.22 μm filter and marked with an E-nr. TheA₂₈₀ is measured using the flow- through as reference. All samples fromthe dialysis and concentrating step are anal- yzed by SDS-PAGE andCoomassie-stained. The purified protein is stored frozen in aliquots anda sheet describing the sample is filed in the “IL5-protein”-folder.

The above-described procedure gives a protein with a purity ofapproximately 90-95%, still containing the His Tag. When sequenced, bothIL5wt and variant IL5.1 gave the expected N-terminal sequences includingthe His Tag.

The purification procedure referred to above has been implemented in thefollowing specific setup:

1) The pooled fractions from the SP-sepharose column are filteredthrough a 0.45 μm filter to remove impurities. A 5-ml HiTrap chelatecolumn (use only dedicated columns) is rinsed with 15 ml water (using asyringe) and then charged with 15 ml 0.1 M NiSO₄ and washed with 15 mlwater. The column is con- nected to the Äkta-system and equilibratedwith 2-3 CV A-buffer. The sample is applied using either the loop orpump - depending on the volume (flow 4 ml/min), the flow-through iscollected and saved for later analysis. Bound protein is eluted using anImi- dazole-gradient going from 0 to 500 mM Imidazole over 20 CV.Fractions of 5 ml are collected. Finally, the column is stripped using 5CV of buffer B2. Buffer A: 0.2 M NaH₂PO₄, 0.5 M NaCl, 10% glycerol, pH5.0 Buffer B1: 0.2 M NaH₂PO₄, 0.5 M NaCl, 0.5 M Imidazole, 10% glycerol,pH 5.0 Buffer B2: 50 mM Na-acetate, 0.5 M NaCl, 0.1 M EDTA, 10%glycerol, pH 4.5 All fractions, flow-through and starting material aretested in dot-blot, all relevant fractions are tested in SDS-PAGE. 2)The purest fractions (as determined by SDS-PAGE) containing IL5 arepooled (save 50 μl for later analysis) and dialyzed twice against 10 Xvolume of PBS, pH adjusted to 6.9. at 6° C., MWCO 12-14 kDa. Thedialysate is filtered through a 0.22 μm filter (save 50 μl for lateranalysis) and A₂₈₀ is measured using filtered dialysis-buffer asreference. The volume before and after dialysis is measured and samplesshowing the dialysis/concentrating step are saved for later analysis bySDS-PAGE (after step 3) 3) After addition of extra NaCl up to a finalconcentration of 400 mM, the dialyzed protein is concentrated usingeither an Amicon apparatus (for volumes larger than 50 ml) or Vivaspinconcen- trating device (for 10-50 ml). In both cases, the membrane issaturated with 10 ml PBS buffer before the sample is applied. The sampleshould be concentrated until a concentration of pre- ferably 1 mg/ml isachieved (as measured by A₂₈₀). The A₂₈₀ is measured using theflow-through as reference. The dialyzed, con- centrated sample isfiltered through a 0.22 μm filter and marked with an E-nr.

All samples from the dialysis and concentrating step are analyzed bySDS-PAGE and Coomassie-stained. The purified protein is stored frozen inaliquots.

Other purification procedures that have been evaluated are:

Zn²⁺-chelate purification: Elution of the protein using an increasingImidazole gradient has proved very efficient as the wt-protein bindsstrongly to the column. The Drosophila supernatant can be directlyapplied and after washing, the IL5wt can be eluted by Imidazole. Thecolumn is charged with 10 CV 10 mM ZnCl₂, and washed with water. The pHof the binding and elution buffers has to be above 6.5 as otherwise theZnCl₂ will precipitate.

Con A affinity chromatography is under investigation. The possibility ofusing the glycosylation present on IL5 as an affinity-tag and elute byapplication of a monosaccharide-analog would be interesting since itcould be applied to the non-His tagged constructs as well.

Removal of Histag:

Removal of the 15 aa His tag (SEQ ID NO: 25) has been performedaccording to suppliers (Unizyme) instructions:

The purified and dialyzed/concentrated His tagged IL5 is de-His taggedby the sequential addition of two enzymes, DAP1 and Glutaminecyclotransferase. DAP1 removes two amino acids from the free N-ter-minus while the QCT The enzyme needs to be activated first: 9 μl HT-DAP1(10 U/ml) is mixed with 9 μl 20 mM cysteamine-HCl. After 5 minincubation at room temperature, a total 108 μl HP-GCT 100 U/ml) and 54μl TAGZyme buffer is added. This must be used within 15 min. Thisportion will digest 1 mg of His tagged protein. The His tagged proteinis mixed with 150 μl activated enzyme and in- cubated at 37° C. for 120min. Samples are withdrawn for SDS-PAGE analysis (10 μl) after 0, 10,30, 60 and 120 min. The samples are put on ice to stop the digestion.Buffers: 1. TAGZyme buffer: 20 mM NaPO₄ buffer, pH 7.5; 150 mM NaCl 2.20 mM Cysteamine-HCl The digested protein (as determined from SDS-PAGEanalysis or N- terminal sequencing) is applied to a 1-ml Ni-chelatecolumn equilibrated in PBS. Everything is collected. The flow-throughfrom the application is saved for later analysis. The column is elutedby addition of 3 CV PBS, fractions of 0.5 ml are collected. The columnis cleaned by washing with 2 CV 0.5 M Imidazole, and fractions are savedfor analysis. All fractions are tested in SDS-PAGE, and fractionscontaining IL5 are pooled and A₂₈₀ is measured using PBS as reference.Finally, the protein is concentrated using a Vivaspin concentratingdevice until a concentration of 1 mg/ml is achieved.

Removal of His tag has been performed in small-scale experiments (0.1-1mg) and has not been up-scaled. It should be noted that removal of thetag requires an unblocked and non-modified N-terminus.

The His tagged protein is incubated with two enzymes, a dipeptidyl aminopeptidase which removes two amino acids at a time and a glutamic acidcyclotransferase which catalyze the conversion of a glutamic acid into apyro-glutamic acid. This conversion blocks further degradation by thedipeptidyl amino peptidase. The digestion mixture is then passed througha chelate column which should retain the enzymes (which are His tagged),contaminating proteins binding to the column and nondegraded orpartially degraded protein. The de-tagged protein passes the column andis collected in the flow-through. After a second digestion with anenzyme that removes the pyro-glutamic acid, the protein is again passedover a chelate-column to remove the second enzyme. It is expected thatthe protein needs to be concentrated again at this final stage.

General Observations:

The pI of UniHis-IL5wt is 9.5 and the optimum pH-value for the proteinseems to be 6.5-7.0 (has not been investigated thoroughly). ANaCl-concentration of 400 mM seems to stabilize the protein duringconcentration.

Example 15

In vitro Screening

The primary in vitro screening will be in the form of an enzyme-linkedimmunosorbent assay (ELISA): A competitive ELISA towards wild-type IL5provides an estimate of the presence of relevant B-cell epitopes in themodified IL5 constructs before introduction thereof into animals.

A conventional ELISA assay can be used to measure titres ofauto-antibodies in the serum of vaccinated animals. Antibodies (bothmono-specific and monoclonal) towards the human as well as towards themurine IL5 are commercially available from R&D Systems, 614 McKinleyPlace NE, Minneapolis, Minn. 55413, USA.

The biological activity of the product and/or the neutralising capacityof induced auto-antibodies can be tested in an IL5 bioassay. Previouslyreported examples of such bioassays are: Assessment of IL5 inducedproliferation of TF1 cells (for human IL5) and assessment of IL5 inducedproliferation of BCL1 cells or B13 B cells (for murine IL5) (Callard &Gearing 1994, Dickason et al., 1994).

The effect on airway responsiveness of the autovaccine can also betested in an in vitro assay wherein the trachea from vaccinated mice areremoved and placed on a hook in an organ bath. The tension of thetrachea after histamine challenge is measured (van Oosterhout et al.,1995).

Consequently, in order to be able to determine the biological activityof recombinant mIL5 (and mIL5 AutoVac) protein samples, a cellularbioactivity assay for murine IL5 is being established. The assay isbased on the ability of the B cell lymphoma line BCL1 to proliferate inresponse to mIL5 added to the culture medium. Two different BCL1 cloneswere obtained from ATCC, BCL1 clone 5B1b (ATCC CRL-1669) and BCL1 cloneCW13.20.3B3 (ATCC TIB-197).

In a typical BCL1 proliferation experiment, the cells are plated incomplete RPMI medium supplemented with fetal calf serum (FCS) inmicrotiter plates and incubated with dilution series of murine IL5.Proliferation of the BCL1 cells is measured by incorporation oftritiated thymidine. Several optimization experiments have beenperformed using dilution series of purchased recombinant mIL5 (R&DSystems) for stimulation. The variable parameters include: incubationintervals, ³H-thymidine pulsing intervals, numbers of cells plated perwell, fetal calf serum (FCS) concentrations and concentrations of addedmIL5. Dose-dependent proliferation of the BCL1 cells with a maximalproliferation of about 3 times the background (BCL1 cells with no mIL5added) has been obtained.

The BCL1 assay has been used to determine the biological activity of thefollowing samples expressed from Drosophila S2 cells and purified asdescribed above: HIS-mIL5wt material (E1320), HIS-mIL5wt material(E1422), HIS-mIL5.1 material (E1396) and an “S2-background-preparation”(E0016). The proliferation in response to one HIS-mIL5wt (E1320)preparation was significantly higher than the proliferation in responseto the “S2-background-preparation”, whereas the mIL5.1 variant and onewild type preparation (E1422) were determined as biologically inactive.

Ongoing work includes inhibition of the BCL1 proliferation withanti-mIL5, and the anti-mIL5 monoclonal antibody TRFK5 is used foroptimization studies. This is done in order to use this assay todetermine the ability of anti-mIL5 antisera from immunised mice toinhibit the biological activity of mIL5.

Example 16

In vivo Models

For measuring the in vivo effect of the autovaccine, well-known animalmodels for asthma exists. Normally, the animal is sensitised with acompound (allergen/antigen) and after challenge with the aerosolisedcompound, broncho-constriction (airway conduction) is measured using abody plethysmograph. The eosinophil cell counts in the BAL fluid arealso determined.

Several of the studies investigating the effect of anti-IL5 mAb's havebeen successfully performed in mice. Against use of the murine modelspeaks the fact the IL5 acts as a B-cell growth factor, renderingpossible interference with the murine antibody response. However, asshown in a study using IL5 knock-out mice, the T-cell dependent antibodyresponse against ovalbumin as well as cytotoxic T-cell developmentappeared normal (Kopf et al., 1996). As the mouse is also the mostpractical and economical model in comparison to guinea pigs or monkeys,the ovalbumin sensitised Bal/c mice model of asthma/airwayhypersensitivity as used by Hamelman et al. (1997) will be used.

If, however, the effect of IL5 on B-cells in the murine model turns outto be a problem, the use of other suitable animal models known in theart will be applied.

Example 17

Preparation of DNA Constructs Encoding Murine IL5 and Variants Thereof

Construction of Variants in pcDNA3.1+:

Insertion of P2 and P30 epitopes into wildtype mIL5 was done by SOE-PCRwith overlapping primers containing the epitope sequences. Wildtype mIL5gene including leader sequence (SEQ ID NO: 63), cloned into pcDNA3.1+with consensus Kozak sequence (obtaining plasmid p815), was used astemplate for the PCR reactions. The resulting fragments were digestedwith NheI and NotI, purified and cloned into p815 was used as templatefor the PCR.

Cloning of Variants into pMT Drosophila Vector With BiP Leader andUNI-His tag:

Wildtype mIL5 was cloned into the pMT Drosophila expression vectorseries (Invitrogen) by generating a PCR fragment with mIL5 specificprimers containing appropriate restriction sites and, in addition,containing sequences encoding a Drosophila Kozak like sequence followedby the Drosophila BiP leader sequence followed by a sequence encoding aUNI-HIS tag (SEQ ID NO: 25) fused to the 5′ end of the sequence encodingmature mIL5. Wildtype mIL5 cDNa sequence was used as template. Theresulting fragment was digested with EcoRI and NotI and was subsequentlycloned into the pMT/V5-HisA vector (Invitrogen). The resulting plasmid(p818) was used for cloning of epitope containing variants into pMT.These were cloned by digesting the variants made in pcDNA3.1+ with SacIand NotI and cloning the resulting fragments into p818.

Cloning of Variants into pAC5:

Wildtype and variants of mIL5 were cloned into the pACS constitutiveDrosophila expression vector by digestion of variants in pMT with EcoRIand NotI and cloning the resulting fragments into the pAC5.1/V5-HisAvector (Invitrogen).

Example 18

Preparation of DNA Constructs Encoding Human IL5 and Variants Thereof

Five lines of plasmids are contemplated containing unmodified IL5 andall or some of the nine IL5 variants. The lines include: 1) human IL5for DNA vaccination in the pCI vector suited for expression in humancells, 2) human IL5 with the BiP leader sequence and a 15 aa His tag(SEQ ID NO: 25, obtained from UNIZYME in Hørsholm, Denmark. The tag istermed “UNI” or “UNI-His tag” herein) in the pMT/V5/HIS vector forinducible expression in Drosophila, 3) as in 2 but without the His tag,4) as in 3 but with murine IL5 and 5) human IL5 with the DAPI leadersequence and the 15 aa HIS tag in the vector pVL1393 for expression inthe baculo-virus system.

Plasmids for DNA-vaccination in the pCI vector:

Name ref # Strain # Epitope hIL5 (pCI) p888 MR#1237 none hIL5.1 (pCI)p889 MR#1238 P2, Loop 3 hIL5.2 (pCI) p890 MR#1239 P30, Loop 1 hIL5.3(pCI) p891 MR#1240 P30, Loop 2 hIL5.4 (pCI) p892 MR#1241 P2, Loop 2hIL5.5 (pCI) p893 MR#1242 P2, Loop 1 hIL5.6 (pCI) p894 MR#1243 P2, Loop3 hIL5.7 (pCI) p895 MR#1244 P30, Loop 3 hIL5.12 (pCI) p896 MR#1245 P30,Loop 3 hIL5.13 (pCI) p897 MR#1246 P2 and P30, Loop 3

Plasmids for human IL5 expression in Drosophila with the UNI-HIS tag andBiP leader sequence in pMT/V5/HIS:

Name Ref # Strain # Epitope hIL5m-UNI-BiP (pMT/V5-HisA) p899 MR#1247none hIL5.1m-UNI-BiP (pMT/V5-HisA) p900 MR#1248 P2, Loop 3hIL5.2m-UNI-BiP (pMT/V5-HisA) p901 MR#1249 P30, Loop 1 hIL5.3m-UNI-BiP(pMT/V5-HisA) p929 MR#1277 P30, Loop 2 hIL5.4m-UNI-BiP (pMT/V5-HisA)p902 MR#1250 P2, Loop 2 hIL5.5m-UNI-BiP (pMT/V5-HisA) p903 MR#1251 P2,Loop 1 hIL5.6m-UNI-BiP (pMT/V5-HisA) p904 MR#1252 P2, Loop 3hIL5.7m-UNI-BiP (pMT/V5-HisA) p905 MR#1253 P30, Loop 3 hIL5.12m-UNI-BiP(pMT/V5-HisA) p906 MR#1254 P30, Loop 3 hIL5.13m-UNI-BiP (pMT/V5-HisA)p907 MR#1255 P2 and P30, Loop 3

Plasmids for human IL5 expression in Drosophila with the BiP leadersequence, but without the UNI-HIS tag in pMT/V5/HIS:

Name Ref # Strain # Epitope hIL5m-BiP (pMT/V5-HisA) p908 MR#1256 nonehIL5.1m-BiP (pMT/V5-HisA) p909 MR#1257 P2, Loop 3 hIL5.2m-BiP(pMT/V5-HisA) p921 MR#1269 P30, Loop 1 hIL5.3m-BiP (pMT/V5-HisA) p922MR#1270 P30, Loop 2 hIL5.4m-BiP (pMT/V5-HisA) p923 MR#1271 P2, Loop 2hIL5.5m-BiP (pMT/V5-HisA) p924 MR#1272 P2, Loop 1 hIL5.6m-BiP(pMT/V5-HisA) p925 MR#1273 P2, Loop 3 hIL5.7m-BiP (pMT/V5-HisA) p926MR#1274 P30, Loop 3 hIL5.12m-BiP (pMT/V5-HisA) p927 MR#1275 P30, Loop 3hIL5.13m-BiP (pMT/V5-HisA) p928 MR#1276 P2 and P30, Loop 3

Plasmids for murine IL5 expression in Drosophila with the BiP leadersequence, but without the 15 aa His tag in pMT/V5/HIS:

Name ref # Strain # Epitope mIL5m-BiP (pMT/V5-HisA) p918 MR#1266 nonemIL5.1m-BiP (pMT/V5-HisA) p919 MR#1267 P2, Loop 3 mIL5.2m-BiP(pMT/V5-HisA) p920 MR#1268 P30, Loop 1

Plasmids for human IL-5 expression in the baculo-virus system with theUNI-HIS tag and DAP1 leader sequence pVL1393 in pVL1393:

Name Ref # Strain # Epitope hIL5m-UNI-DAP1 (pVL1393) p916 MR#1264 nonehIL5.1m-UNI-DAP1 (pVL1393) p917 MR#1265 P2, Loop 3

Example 19

DNA Immunization Studies

Generation of Vectors Encoding mIL5wt, mIL5.1 and mIL5.5 With KozakSequences for DNA Vaccination Experiments:

DNA fragments encoding mIL5wt, mIL5.1 and mIL5.5 including the naturalleader sequence (SEQ ID NO: 63) were inserted into pcDNA3.1 thusyielding new plasmids p521, 522, and p523. In 10 order to enhanceexpression of cDNA in mammalian cells, Kozak concensus sequences wereinserted upstream of the coding sequences using PCR technology. PCRreactions were performed using p521, p522 and p523 as templates and aforward primer encoding the Kozak sequence immediately upstream of themIL5 leader start codon. Purified PCR products were cloned intopcDNA3.1+ vector using restriction endonucleases BamHI and NotI. Theresulting plasmids p815, p816 and p817, respectively, were verified byDNA sequencing. All other plasmids used for DNA vaccination experimentswere constructed using the p521 plasmid as starting material.

In vitro Translation of DNA Vaccination Plasmids Using Promega Kit:

A commercial kit using rabbit reticulocyte extract to generate 25 invitro translated protein product plasmid DNA, has previously beensuccessfully used in our lab to monitor protein expression from pcDNAplasmid encoding e.g ovalbumin cDNA. Murine IL5 DNA vaccination plasmidswere added to the kit reagents according to the standard procedure.However, several attempts to detect expressed mIL5 material onautoradiograms failed whereas positive controls worked. Results from COScell transfections and DNA vaccination:shows that the gene products areexpressed, so we did not investigate these technical problems further.

Transient Transfection of COS Cells With DNA Vaccination Plasmids toDetermine Expression Levels:

In order to monitor the transfection/expression efficiency of theplasmids used for DNA vaccination experiments, a transient transfectionassay using COS cells was established. COS cells were trypsinized andplated in DMEM medium supplemented with 10% FCS in T25 culture flasks.The cells were transfected at day 2 using the Dotap kit (RocheDiagnostics) and harvested at day 5. Culture supernatant, whole celllysate and membrane enriched preparations were tested in Westernblotting to detect anti-mIL5 reactive expression product. The anti-mIL5reactive product in the cell preparations consistently migrated as 2-3separate bands of 21-28 kD in SDS-PAGE, whereas the MW of the mIL5monomer used as standard (expressed in bacculovirus, R&D Systems) isonly 15-18 kD. Using non-denaturating circumstances, the 21-28 kDsubstances form dimers so we believe the material is mIL5, possibly inseveral differently glycosylated forms. DNA vaccination results (seebelow) clearly support this conclusion.

DNA Vaccination of Mice Using Murine IL5 AutoVac Constructs:

A DNA vaccination study was performed in order to investigate whetherantibody responses specific for murine IL5 can be induced by immunisingmice with naked plasmid DNA encoding 8 different murine IL5 mutants.Since IL5 previously has been reported to play a role in B celldifferentiation, it is essential to demonstrate that anti-mIL5autoantibodies can be generated in mice and B cell tolerance to mIL5 canbe broken.

The general setup of the DNA vaccination experiments use either C3H/Henmice (H-2^(k)) or Balb/cA mice (H-2^(d)), 6-8 weeks old divided intogroups of 5 mice each. At days 0, 14, 28, 42, 62 and 76 the mice wereanaestesized using hypnorm/dormicum s.c. and injected with expressionplasmids encoding ovalbumin (control), mIL5wt (wild type), or the mIL5variants to be tested. The DNA material was prepared using endofreeGigaPrep kits (Qiagen) and dissolved at 1 μg/ml in 0.15 M NaCl or 0.15 MNaCl containing 0.1% bupivacaine. 100 μl material was injected i.d. ineach mouse at the lower back distributed at two injection sites.Prebleeds were obtained at day minus 2, and the test bleedings wereobtained at weeks 3, 5, 8 and 11. Sera were isolated by centrifugationand stored at −20° C. until testing in ELISA for reactivity againstpurified ovalbumin and mIL5 proteins.

A Typical result of a DNA vaccination experiment is shown in FIG. 4.According to the general setup described above, 40 Balb/cA mice wereimmunized with ovalbumin control plasmid, mIL5wt encoding plasmid orplasmids encoding the mIL5 AutoVac variants mIL5.1 or mIL5.5. In thisexperiment, 9 out of 9 mice immunized with ovalbumin encoding plasmiddeveloped anti-oval-bumin antibodies, whereas no anti-ovalbumin responsewas induced in mice receiving the mIL5 wild type or mIL5 variantencoding DNA. Injection of mIL5wt encoding plasmid did not give raise toan anti-mIL5 response, whereas the B cell tolerance to mIL5 was brokenin 4 out of 10 mice immunized with mIL5.1 plasmid and 7 out of 9 miceimmunized with mIL5.5 encoding plasmid DNA.

The main result of the whole series of DNA vaccination experiments issummarized in the table below. The number of responders within animmunisation group differs between the different mIL5 AutoVac constructsand is dependent on the mouse strain. Clearly, the mIL5.2 AutoVacconstruct is superior to the other variants, being able to induceanti-mIL5 antibody responses in both mouse strains with a penetrance of100%.

This plasmid (p820) also gave the highest expression levels in the COStransfection assay.

Another example to emphasize is the apparent MHC restriction seen whenusing mIL5.4 encoding plasmid DNA as immunogen. Whereas only 1/10C3H/Hen mice responds to the DNA vaccine, 9 out of 10 Balb/cA mice areresponders. The opposite phenomenon (although not quite as pronounced)is seen with the mIL5.6 construct. The mIL5.2 DNA vaccine, however, seemto be promiscuously immunogenic.

OVAwt-pVax mIL5wt-pcDNA mIL5.1-pcDNA mIL5.2-pcDNA mIL5.4-pcDNA Balb/cA28/28 0/28 4/10  9/10 9/10 C3H/Hen 29/29 0/30 3/10 10/10 1/10mIL5.5-pcDNA mIL5.6-pcDNA mIL5.7-pcDNA mIL5.12-pcDNA mIL5.13-pcDNABalb/cA  7/9 0/10 2/10 0/10 0/10* C3H/Hen 5/10 6/10 2/10 2/10 2/10*Summary of the result of DNA vaccination of 280 mice. 6 mice died duringthe experiment for reasons not connected to the effects of the DNAvaccination. The number of responders (with high or intermediateanti-mIL5 titers) is shown in relation to the total number of micewithin each immunization group. *bleedings obtained at day 55. All theother bleedings were obtained at day 77.

Another feature to mention is the tendency of mIL5 variants with theforeign T helper epitope inserted in mIL5 loop1 to be stronger DNAvaccination immunogens than variants with the T helper epitope insertedin loop 3. This could be due to the relatively high expression levels.The only loop 2 variant tested, mIL5.4-pcDNA is only a strong immunogenin the Balb/cA strain, as mentioned above.

Further Characterization of the Antibody Responses Induced by DNAVaccination:

ELISA experiments were set up in order to determine whether antibodiesspecific for the inserted T helper epitope could be detected inanti-mIL5 positive mice. For each immunisation group, sera fromanti-mIL5 positive mice were pooled and tested for reactivity against P2or P30 peptides which had been immobilised in AquaBind microtiterplates. Antisera induced by DNA vaccination against mIL5.2 in both mousestrains clearly contained reactivity against the inserted P30, whereasnone of the other antisera were reactive with P2 or P30. This isprobably connected to the higher antibody titers and penetrance that isgenerally observed with the mIL5.2 DNA vaccination construct. It shouldbe mentioned that using this ELISA setup we were able to detect anti-P2reactivity in antisera induced against mIL5.1.

The positive anti-mIL5 antiserum pools from the DNA vaccinated mice werealso tested in a competive ELISA for their ability to inhibit theinteraction between soluble native murine IL5 and monoclonal antibodiesTRFK4 or TRFK5, which are both neutralizing antibodies. Dilution seriesof anti-mIL5 antiserum pools were preincubated with soluble native mIL5and the sample was added to ELISA plates coated with catching antibodyTRFK5. Bound murine IL5 (which was not absorbed by the antisera) wasnext visualised using layers of biotinylated TRFK4 and subsequentlyhorse radish peroxidase labeled streptavidin. Not all the anti-mIL5positive antisera induced by DNA vaccination could inhibit theinteraction between soluble mIL5 and TRFK4 or TRFK5. The antiserum withthe highest TRFK4/5 inhibiting capability was from C3H/Hen miceimmunized with mIL5.2 encoding DNA. It has not been tested whether theobserved differences in inhibition is a direct measure of titerdiffernces or it is connected to the fine specificity of the differentantisera. Most likely, it is a combination of these two factors.

Animal Model of Eosinophilia in mIL5 AutoVac DNA Immunized Mice:

40 DNA vaccinated mice were chosen for testing in an animal model ofeosinophilia: 10 Balb/cA mice immunized with mIL5wt DNA, 10 Balb/cA miceimmunized with mIL5.2 DNA, 10 C3H/Hen mice immunized with mIL5wt DNA and10 C3H/Hen mice immunized with mIL5.2 DNA. A sensitization/challengingregimen with ovalbumin to induce eosinophilia was given to in each ofthese mice. The mice were sensitized with subcutaneous injections of 50μg ovalbumin (OVA) in 0.9% saline mixed 1:1 with Adjuphos once per weekfor three weeks. Four days after the last OVA sensitization the micewere challenged intranasally with 12.5 μg OVA in 0.9% saline every otherday for a total of 3 challenges. Bronchoalveolar lavage fluid (BALF) wascollected one day after the last sensitization by cannulating thetracheae and washing the airway lumina with 1 ml PBS.

Approximately 30,000-60,000 BALF cells were spun unto slides at 1,500rpm for 20 minutes. The slides were dried overnight and stained for 2.5minutes with May-Grunwald stain (Sigma), washed for 4 minutes in trisbuffered saline, stained for 20-30 minutes with Geimsa stain (1:20 withddH2O; Sigma) and briefly rinsed with ddH2O. Stained slides were driedovernight and cell types were identified using light microscopy.Approximately 100-200 cells were counted per slide and 3 slides werecounted per mouse. The eosinophil counts were expressed as the number ofeosinophils per 100 cells counted. In mIL5.2 DNA vaccinated C3H/Henmice, the induction of lung eosinophilia was significantlydown-regulated compared to the wild type mIL5wt DNA vaccinated group(mIL5.2 DNA: 14.6±8.9 eosiophils per 100 cells; mIL5wt DNA: 51.1±9.9eosinophils per 100 cells). However, in the Balb/cA strain, there was nosignificant difference in eosinophil counts between the immunizationgroups (mIL5.2 DNA: 23.3±6.8 eosinophils per 100 cells; mIL5wt DNA:27.7±9.3 eosinophils per 100 cells). A possible explanation is thatBalb/cA mice are only weakly susceptible to the model. This is supportedby anti-ovalbumin ELISA data showing that one week before the BALFcollection the antiovalbumin titers in serum from the Balb/cA mice werelower than in serum from C3H/Hen. The Balb/cJ substrain is reported tobe susceptible to the OVA sensitization/challenge model.

Example 20

Protein Vaccination Study

Balb/c J mice were immunized with murine IL5 (mIL5) protein andsubjected to an ovalbumin intranasal model that induces eosinophils inthe lungs of treated mice. Both the UniHis-mIL5 and the UniHis-mIL5.1proteins induced antibodies that cross-react with mIL-5 made in sf9cells from R&D Systems. The eosinophilia model induced high numbers ofeosinophils in the OVA control group and the UniHis-mIL5.1 groups, whilethe numbers of eosinophils were reduced in both the PBS group and theUniHis-mIL5 group. -This result led us to believe that the groups mayhave been mixed.

Materials & Methods:

UniHis-mIL-5 E1320 & E01397 UniHis-mIL-5.1 E01337 & E01396

Immunizations:

6-8 week old female Balb/c J (M&B) mice were immunized with either 1)nothing, 2) PBS, 3) UniHis-mIL5, or 4) UniHis-mIL-5.1 in CompleteFreund's Ajuvant (CFA; Sigma) and boosted 3 times at three weekintervals with antigen in Incomplete Freund's Adjuvant (IFA; Sigma).Sera was collected and tested in an ELISA 10 days after each boost.

ELISAs:

Anti-UniHis-mIL5 ELISA:

Sera were obtained at days 32 (bleed 1) and 54 (bleed 2) after 2 and 3immunizations, respectively. Polystyrene microtiter plates (Maxisorp,Nunc) were coated with purified HIS-mIL5wt (0.1 μg/well, E1320). Thereactivities of diluted sera added to the wells were visualised using agoat anti-mouse secondary antibody. OD490 readings of control sera frommice immunized with PBS in Freunds adjuvans were subtracted from theOD490 readings of the test samples.

Anti-mIL5 ELISA:

Sera were obtained at day 75 (bleed 3). Polystyrene microtiter plates(Maxisorp, Nunc) were coated with purchased mIL5 (0.1 μg/well, R&D cat.no. 405-ML). The reactivities of 1:1000 diluted sera added to the wellswere visualised using a goat anti-mouse secondary antibody. Thereactivity of TRFK5 (2 μg/ml) was visualised using a rabbit anti-ratsecondary antibody.

Competitive ELISA:

Dilutions of antisera were preincubated with soluble IL5 for 1 hour andadded to polystyrene microtiter plates (Maxisorp, Nunc) which werecoated with catching antibody TRFK5. Bound mIL5 was visualised usingbiotinylated TRFK4 and a HRP labelled goat anti-mouse secondaryantibody.

Anti-P2 ELISA:

Pools of antisera from HIS-mIL5wt, HIS-mIL5.1 or PBS immunised mice weretested for reactivity against P2 peptide in ELISA. Specializedmicrotiter plates (Aquabind, M&E Biotech) were coated with 0.5 μg/wellsynthetic P2 peptide. The reactivities of diluted sera added to thewells were visualised using a HRP labelled goat anti-mouse secondaryantibody (1:2000, Dako).

Anti-UniHis ELISA:

Pools of antisera from HIS-mIL5wt, HIS-mIL5.1 or PBS immunised mice weretested for reactivity against HIS-tag peptide (UNIZYME) in ELISA.Specialized microtiter plates (AquaBind, M&E Biotech) were coated with0.5 μg/well synthetic HIS-tag peptide. The reactivities of diluted seraadded to the wells were visualised using a HRP labelled goat anti-mousesecondary antibody (1:2000, Dako).

Anti-S2 Background Protein ELISA:

Pools of antisera from HIS-mIL5wt; HIS-mIL5.1 or PBS immunised mice weretested for reactivity against S2 background preparation in ELISA.Polystyrene microtiter plates (Maxisorp, Nunc) were coated with 0.1μg/well S2 background preparation. The reactivities of diluted seraadded to the wells were visualised using a HRP labelled goat anti-mousesecondary antibody (1:2000, Dako).

Anti-BSA ELISA:

Pools of antisera from HIS-mIL5wt, HIS-mIL5.1 or PBS immunised mice weretested for reactivity against BSA in ELISA. Polystyrene microtiterplates (Maxisorp, Nunc) were coated with 10 μg/well BSA (Intergen). Thereactivities of diluted sera added to the wells were visualised using aHRP labelled goat antimouse secondary antibody (1:2000, Dako).

Eosinophilia Model:

Balb/c J mice were sensitized with subcutaneous injections of 50 μgovalbumin (OVA) in 0.9% saline mixed 1:1 with Adjuphos as alum adjuvant.OVA immunizations were repeated once per week for four weeks. One weekafter the last OVA sensitization, the mice were challenged with 12.5 μgOVA in 0.9% saline intranasal every other day for a total of 3challenges. Bronchoalveolar lavage fluid (BALF) was collected one dayafter the last sensitization by cannulating the tracheae and washing theairway lumina with 1 ml 0.9% saline, or PBS.

BAL Staining:

Approximately 30,000-60,000 BALF cells were spun unto slides at 1,500rpm for 20 minutes. The slides were dried overnight and stained for 2.5minutes with May-Grunwald stain (Sigma), washed for 4 minutes in TBS,stained for 20-30 minutes with Giemsa stain (1:20 with ddH₂O; Sigma) andbriefly rinsed with ddH₂O. Stained slides were dried overnight and celltypes were identified using light microscopy. Approximately 100-200cells were counted per slide and 3 slides were counted per mouse.

Results:

Detection of Anti-mIL5 Antibodies:

A series of ELISA experiments were performed in order to investigatewhether antibody responses specific for murine IL5 were induced in miceimmunized with HIS-mIL5wt and HIS-mIL5.1 protein material. First, it wasdetermined if antibodies against the HIS-mIL5wt immunization materialwere elicited by testing dilutions of antisera from individual mice onELISA plates coated with the HIS-mIL5wt material. It was found thatalready by bleed one, all mice had developed high-titered antibodyresponses against the HIS-mIL5wt material (E1320, expressed fromDrosophila S2 cells and purified) which was estimated to beapproximately 95% pure.

This result is not a firm confirmation that the antisera cross-reactswith murine IL5. In this setup, reactivities would also be detectedagainst impurities from the Drosophila S2 cells, the S2 medium (whichcontain e.g. BSA from fetal 10 calf serum, the HIS-tag as well asdenatured mIL5 B cell epitopes. To demonstrate, that the antibodiesinduced contain reactivities against native murine IL5, the sera weretested in ELISA plates coated with mIL5 purchased from R&D systems. Thismaterial (R&D cat. no. 405-ML) is biologically active, contains noHIS-tag, is expressed in the bacculovirus Sf21 system, is also very pure(97%), and can be purchased free of carrier-protein (BSA). Pooled serafrom both immunisation groups reacted with the purchased mIL5 coated onELISA plates, whereas sera from PBS immunised mice did not. This wasshown when testing sera from bleed 3 obtained at day 75, 11 days afterthe 4^(th) immunization, but also sera from bleed 1 and 2 reacts withthe purchased mIL5 in a similar setup. In order to exclude signals fromcross-reaction with the BSA carrier, the experiments were repeated forbleeds 1 and 2 using carrier-free versions of the purchased mIL5material and BSA-free ELISA buffers, and still high anti-mIL5 responsesare seen.

To further confirm that the induced antisera cross-react with nativemIL5, a competitive ELISA was set up. This ELISA tests the ability ofthe different antisera to inhibit the interaction between soluble nativemurine IL5 and monoclonal antibodies TRFK4 or TRFK5, which are bothneutralizing antibodies. Dilution series of antiserum pools werepreincubated with soluble native mIL5 and the samples were added toELISA plates coated with catching antibody TRFK5. Bound murine IL5(which was not absorbed by the antisera) was next visualised usinglayers of biotinylated TRFK4 and subsequently horseradish peroxidaselabeled streptavidin. An anti-mIL5 positive and an anti-mIL5 negativeantiserum from DNA vaccinated mice were included as controls. It wasdemonstrated that antisera from both HIS-mIL5wt and HIS-mIL5.1 immunizedmice could inhibit the interaction between soluble mIL5 and TRFK4 orTRFK5.

Based on the above-referenced it is concluded that mIL5 specificautoantibodies are induced in mice immunized with either the HIS-mIL5wtor the HIS-mIL5.1 protein preparations (in 100% of the mice tested). Inother words, B cell tolerance to mIL5 can be broken using recombinantHIS-tagged versions of both wild type and AutoVac murine IL5. Aplausible explanation for the observation that B cell tolerance isbroken to the wild type protein is that the HIS-tag in these micefunctions as a “foreign” immunogenic T helper epitope. Anotherexplanation could be that the administration of Complete Freund'sAdjuvant breaks B cell tolerance to mIL5. These hypotheses can be testedusing non-HIS tagged antigens and/or alternative, less strong adjuvantssuch as AdjuPhos.

Further Characterization of the Antibody Responses in Mice ImmunizedWith mIL5 AutoVac Proteins:

ELISA experiments were set up in order to determine whether antibodiesspecific for the inserted T helper epitope could be detected in serafrom mIL5 protein immunised mice. For each immunisation group, antisera(bleed 2) were pooled and tested for reactivity against synthetic P2peptide which had been immobilised in AquaBind microtiter plates.Anti-HIS-mIL5.1 antiserum contained reactivity against the inserted P2peptide, whereas neither anti-HIS-mIL5wt or anti-PBS/CFA reacted withthe peptide.

It was also tested whether the the anti-HIS-mILwt and anti-HIS-mIL5.1antisera contained reactivity against the 15-mer HIS-tag (UNIZYMEHIS-tag, SEQ ID NO: 25) that is fused to the N-terminal of both the wildtype and AutoVac mIL5 proteins. The peptide was synthesized andcovalently immobilized in AquaBind microtiter plates, and pooledantisera from each immunization group (bleeds 1, 2 and 3) were testedfor reactivity against the bound peptide. Antisera from all proteinimmunized mice reacted with the synthetic HIS-tag peptide.

It was also tested whether the anti-HIS-mIL5wt and anti-HIS-mIL5.1antisera was reactive with components from the S2 Drosophila cells orculture medium. ELISA plates coated with BSA (a major medium component)or S2-background preparation (generated by subjecting culturesupernatant from Her2 expressing Drosophila S2 cells to a purificationscheme similar to that of the mIL5 purification procedure). The resultsof these analyses demonstrated that whereas the anti-BSA responses werevery low, the reactions with the S2-background material were pronounced.

Eosinophil Counts in BALF:

To determine if the anti-IL5 antibodies in vaccinated mice coulddown-regulate the in vivo activity of IL5, we induced IL5-dependenteosinophilia in the lungs of the vaccinated mice. Eosinophils wereinduced by challenging sensitized mice with OVA intranasally. Highnumbers of eosinophils were induced in control OVA mice and micevaccinated with UniHis-mIL5.1, but not in Uni-His-mIL5 or PBS vaccinatedmice. The discrepancy of eosinophil numbers between control groups (OVAand PBS) and experimental groups (UniHis-mIL5 and UniHis-mIL5.1), andthe positive results from the DNA vaccinated mice reported above, led usto believe that the groups may have been switched. However, no attemptsto demonstrate a switch supported this interpretation. The proteinvaccinations are being repeated in an identical setup to clarify thiscontroversy.

Discussion:

The ability of both the UniHis-mIL5 and UniHis-mIL5.1 proteins to induceantibodies that cross-react with wildtype murine IL5 was clearlydemonstrated. Whether the ability of the UniHis-mIL5 protein to bypassimmunological tolerance is due to the UniHis-tag, or some other reason(e.g. CFA adjuvant) remains to be clarified. It was surprising to seethat only the Uni-His-mIL5 construct was able to down-regulate theendogenous in vivo activity of mIL5 in an eosinophilia model. Thisinability of antisera generated from UniHis-mIL5.1 protein vaccinationto inhibit eosinophilia, and its ability to inhibit eosinophilia via DNAvaccinations suggests that a technical mistake may have occurred in thisexperiment. This is also supported by the unusual finding of PBSvaccination inhibiting eosinophilia. This most likely explanation isthat these two groups (PBS and UniHis-mIL5.1) were switched.

LIST OF REFERENCES

Akutsu I. et al. ,1995, Immunol. Lett., 45: 109-116. Alexander A. G. etal., 1994, Thorax, 49(12): 1231-1233.

Azuma C. et al., 1986, Nucleic Acid Res. 1986, 14(22): 9149-9158.

Barata L. T. et al., 1998, J. Allergy and Clin. Immunol, 101: 222-230.

Baumann M. A. et al., 1997, Methods, 11: 88-97

Callard R. E. & Gearing A. J. H., ‘IL-5’, Cytokine Facts Book 994,Academic Press.

Campbell H. D. et al., 1988, Eur. J. Biochem., 174: 345-352.

Chand N. et al., 1992, Eur. J. Immunol., 211: 121-123.

Coeffier E. et al., 1994, Br. J. Pharmacol., 113(3): 749-56.

Coffman R. L. et al., 1989, Science, 245: 308-310.

Corrigan C. J. & Kay A. B., 1996, Eur. Resp. J., 9, suppl. 22: 72s-78s.

Cousins D. J. et al., 1994, Am. J. Resp. Crit. Care. Med., 150: S50-S53.

Danzig M. et al., 1997, Allergy, 52(8): 787-794.

Dickason R. R. et al., 1994, Cytokine, 6(6): 647-656.

Dickason R. R. et al., 1996a, Nature, 379: 652-655.

Dickason R. R. et al., 1996b, J. Mol. Med., 74(9), 535-546

Egan R. W. et al., 1995, Int. Arch. Allergy Immunol., 107: 321-322.

Foster P. S. et al., 1996, J. Exp. Med., 183: 195-201.

Graber P. et al., 1993, Eur. J. Biochem., 212(3): 751-755.

Graber P. et al., 1995, J. Biol. Chem., 270(26): 15762-15769.

Hamelmann E. et al., 1997, Am. J. Crit. Care Med., 155(3): 819-825.

Huston M. M. et al., 1996, J. Immunol., 156(4): 1392-1401.

Karlen S. et al., 1998, Int. Rev. Immunol., 16(3-4): 227-247.

Kodama S. et al., 1993, Eur. J. Biochem., 211(3): 903-908.

Kopf M. et al., 1996, Immunity, 4: 15-24.

Kung T. T. et al., 1995, Am. J. Respir. Cell. Mol. Biol., 13: 360-365.

Lee N. A. et al., 1997a, J. Immunol., 158: 1332-1344.

Lee J. J. et al., 1997b, J. Exp. Med. 1997b, 185(12): 2143-2156.

Lopez A. F. et al., 1992, Immunology Today, 13: 495-500.

Mauser P. J. et al., 1993, Am. Rev. Respir. Dis., 148: 1623-1627.

Mauser P. J. et al., 1995, Am. J. Respir. Crit. Care Med., 152(2):467-472.

Milburn M. V. et al., 1993, Nature, 363: 172-176.

Mori A. et al., 1997, J. Allergy Clin. Immunol., 100(6) Pt 2: S56-64.

Moxham J. & Costello J. F., ‘Respiratory diseases’, chapt. 14, Textbookof Medicine, Churchill Livingstone 1990, Ed. Souhami R. L. and Moxham J.

Nagai H. et al., 1993a, Ann. N.Y. Acad.Sci., 91-96.

Nagai H. et al., 1993b, Life Sciences, 53: PL 243-247.

Ohashi Y. et al., 1998, Scand. J. Immunol, 47: 596-602.

Ortega D. & Busse W. W., ‘Asthma: Pathogenesis and treatment’, chapt.28, Allergy, W. B. Saunders Company 1997, Ed. Kaplan A. P.

Proudfoot A. E. et al., 1990, Biochem J., 270(2): 357-361.

Proudfoot A. E. et al., 1996, J. Protein Chem., 15(5): 491-499.

Rose K. et al., 1992, Biochem J, 286(Pt 3): 825-828.

Sanderson C. J., 1992, Blood, 79(:12): 3101-3109.

Sher A. et al., 1990, J. Immunol., 145: 3911-3916.

Takatsu K. et al., Interleukin-5, Growth Factors and Cytokines in Healthand Disease 1997, vol.2A, JAI Press Inc., Ed. Leroith D. & Bondy C.

Tanabe T. et al., 1987, J. Biol. Chem., 262: 16580-16584.

Tavernier J. et al., 1989, DNA, 8(7), 491-501.

Tominaga A. et al., 1990, J. Immunol., 144(4): 1345-1352.

Tominaga A. et al., 1991, J. Exp. Med., 173(2): 429-437.

Underwood D. C. et al., 1996, Am. J. Resp. Crit. Care Med., 154:850-857.

van Oosterhout A. J. M. et al., 1993, Am. Rev. Resp. Dis., 147: 548-552.

van Oosterhout A. J. M. et al., 1995, Am. J. Respir. Crit. Care Med.,151: 177-183.

Villinger F. et al., 1995, J. Immunol., 155: 3946-3954.

Wang P. et al., 1998, J. Immunol., 160: 4427-4432.

Yamaguchi Y. et al., 1991, Blood, 78(10): 2542-2547.

65 1 115 PRT Homo sapiens DISULFID (44) Interchain disulphide bond toCys-86 in SEQ ID NO1 1 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val LysGlu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile AlaAsn Glu Thr Leu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln LeuCys Thr Glu Glu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln ThrVal Gln Gly Gly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu IleLys Lys Tyr Ile Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Glu Glu ArgArg Arg Val Asn Gln Phe 85 90 95 Leu Asp Tyr Leu Gln Glu Phe Leu Gly ValMet Asn Thr Glu Trp Ile 100 105 110 Ile Glu Ser 115 2 126 PRT ArtificialSequence Description of Artificial SequenceHuman IL5 modified bysubstitution with tetanus toxoid P2 epitope 2 Ile Pro Thr Glu Ile ProThr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr HisArg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile Pro Val Pro ValHis Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 Phe Gln Gly Ile GlyThr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 Val Glu Arg Leu PheLys Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 Gly Gln Lys LysLys Cys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile 85 90 95 Gly Ile Thr GluLeu Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln 100 105 110 Glu Phe LeuGly Val Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120 125 3 118 PRTArtificial Sequence Description of Artificial SequenceHuman IL5 modifiedby substitution with tetanus toxoid P2 epitope 3 Ile Pro Thr Glu Ile ProThr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr HisArg Thr Leu Leu Ile Ala Asn Glu Thr Leu Gln 20 25 30 Tyr Ile Lys Ala AsnSer Lys Phe Ile Gly Ile Thr Glu Leu Cys Thr 35 40 45 Glu Glu Ile Phe GlnGly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln 50 55 60 Gly Gly Thr Val GluArg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys 65 70 75 80 Tyr Ile Asp GlyGln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val 85 90 95 Asn Gln Phe LeuAsp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr 100 105 110 Glu Trp IleIle Glu Ser 115 4 124 PRT Artificial Sequence Description of ArtificialSequenceHuman IL5 modified by substitution with tetanus toxoid P2epitope 4 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr LeuAla 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr GluGlu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Gln Tyr Ile LysAla Asn 50 55 60 Ser Lys Phe Ile Gly Ile Thr Glu Leu Val Glu Arg Leu PheLys Asn 65 70 75 80 Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys LysLys Cys Gly 85 90 95 Glu Glu Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr LeuGln Glu Phe 100 105 110 Leu Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser115 120 5 124 PRT Artificial Sequence Description of ArtificialSequenceHuman IL5 modified by substitution with tetanus toxoid P2epitope 5 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr LeuAla 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr GluGlu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln GlyGly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys TyrIle Asp 65 70 75 80 Gly Gln Lys Lys Lys Gln Tyr Ile Lys Ala Asn Ser LysPhe Ile Gly 85 90 95 Ile Thr Glu Leu Arg Val Asn Gln Phe Leu Asp Tyr LeuGln Glu Phe 100 105 110 Leu Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser115 120 6 126 PRT Artificial Sequence Description of ArtificialSequenceHuman IL5 modified by substitution with tetanus toxoid P2epitope 6 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr LeuAla 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr GluGlu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln GlyGly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys TyrIle Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg ValAsn Gln Phe 85 90 95 Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn ThrGln Tyr Ile 100 105 110 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu LeuGlu Ser 115 120 125 7 132 PRT Artificial Sequence Description ofArtificial SequenceHuman IL5 modified by substitution with tetanustoxoid P30 epitope 7 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys GluThr Leu Ala 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala AsnGlu Thr Leu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu CysThr Glu Glu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr ValGln Gly Gly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile LysLys Tyr Ile Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Phe Asn Asn Phe ThrVal Ser Phe Trp Leu 85 90 95 Arg Val Pro Lys Val Ser Ala Ser His Leu GluArg Arg Val Asn Gln 100 105 110 Phe Leu Asp Tyr Leu Gln Glu Phe Leu GlyVal Met Asn Thr Glu Trp 115 120 125 Ile Ile Glu Ser 130 8 124 PRTArtificial Sequence Description of Artificial SequenceHuman IL5 modifiedby substitution with tetanus toxoid P30 epitope 8 Ile Pro Thr Glu IlePro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser ThrHis Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Phe 20 25 30 Asn Asn Phe ThrVal Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala 35 40 45 Ser His Leu GluCys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu 50 55 60 Glu Ser Gln ThrVal Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn 65 70 75 80 Leu Ser LeuIle Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly 85 90 95 Glu Glu ArgArg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 Leu GlyVal Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120 9 130 PRT ArtificialSequence Description of Artificial SequenceHuman IL5 modified bysubstitution with tetanus toxoid P30 epitope 9 Ile Pro Thr Glu Ile ProThr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr HisArg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile Pro Val Pro ValHis Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 Phe Gln Gly Ile GlyThr Leu Glu Ser Gln Phe Asn Asn Phe Thr Val 50 55 60 Ser Phe Trp Leu ArgVal Pro Lys Val Ser Ala Ser His Leu Glu Val 65 70 75 80 Glu Arg Leu PheLys Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly 85 90 95 Gln Lys Lys LysCys Gly Glu Glu Arg Arg Arg Val Asn Gln Phe Leu 100 105 110 Asp Tyr LeuGln Glu Phe Leu Gly Val Met Asn Thr Glu Trp Ile Ile 115 120 125 Glu Ser130 10 132 PRT Artificial Sequence Description of ArtificialSequenceHuman IL5 modified by substitution with tetanus toxoid P30epitope 10 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr LeuAla 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr GluGlu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln GlyGly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys TyrIle Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg ValAsn Gln Phe 85 90 95 Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn ThrPhe Asn Asn 100 105 110 Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys ValSer Ala Ser His 115 120 125 Leu Glu Glu Ser 130 11 141 PRT ArtificialSequence Description of Artificial SequenceHuman IL5 modified bysubstitution with tetanus toxoid P2 and P30 epitopes 11 Ile Pro Thr GluIle Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu SerThr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile Pro ValPro Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 Phe Gln GlyIle Gly Thr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 Val Glu ArgLeu Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 Gly GlnLys Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 85 90 95 Ile ThrGlu Leu Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 LeuGly Val Met Asn Thr Phe Asn Asn Phe Thr Val Ser Phe Trp Leu 115 120 125Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Glu Ser 130 135 140 12 113PRT Mus musculus DISULFID (42) Interchain disulphide bond to Cys-84 inSEQ ID NO12 12 Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu AlaLeu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr MetArg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly GluIle Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asp Gln Thr Val Arg Gly GlyThr Val Met 50 55 60 Arg Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr IleAsp Arg Gln 65 70 75 80 Glu Lys Lys Cys Gly Glu Glu Arg Arg Arg Thr ArgGln Phe Leu Asp 85 90 95 Tyr Leu Gln Glu Phe Leu Gly Ser Met Asn Thr AlaAla Ile Ile Glu 100 105 110 Gly 13 124 PRT Artificial SequenceDescription of Artificial SequenceMurine IL5 modified by substitutionwith tetanus toxoid P2 epitope 13 Met Glu Ile Pro Met Ser Thr Val ValLys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His GlnLeu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asp GlnThr Val Arg Gly Gly Thr Val Met 50 55 60 Arg Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Glu Lys Lys Cys Gln Tyr IleLys Ala Asn Ser Lys Phe Ile Gly Ile 85 90 95 Thr Glu Leu Arg Arg Thr ArgGln Phe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 Leu Gly Ser Met Asn ThrAla Ala Ile Ile Glu Gly 115 120 14 116 PRT Artificial SequenceDescription of Artificial SequenceMurine IL5 modified by substitutionwith tetanus toxoid P2 epitope 14 Met Glu Ile Pro Met Ser Thr Val ValLys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Gln Tyr Ile 20 25 30 Lys Ala Asn Ser Lys Phe Ile GlyIle Thr Glu Leu Cys Ile Gly Glu 35 40 45 Ile Phe Gln Gly Leu Asp Ile LeuLys Asp Gln Thr Val Arg Gly Gly 50 55 60 Thr Val Met Arg Leu Phe Gln AsnLeu Ser Leu Ile Lys Lys Tyr Ile 65 70 75 80 Asp Arg Gln Glu Lys Lys CysGly Glu Glu Arg Arg Arg Thr Arg Gln 85 90 95 Phe Leu Asp Tyr Leu Gln GluPhe Leu Gly Ser Met Asn Thr Ala Ala 100 105 110 Ile Ile Glu Gly 115 15122 PRT Artificial Sequence Description of Artificial SequenceMurine IL5modified by substitution with tetanus toxoid P2 epitope 15 Met Glu IlePro Met Ser Thr Val Val Lys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser AlaHis Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val ProThr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly LeuAsp Ile Leu Lys Asp Gln Gln Tyr Ile Lys Ala Asn Ser Lys 50 55 60 Phe IleGly Ile Thr Glu Leu Val Met Arg Leu Phe Gln Asn Leu Ser 65 70 75 80 LeuIle Lys Lys Tyr Ile Asp Arg Gln Glu Lys Lys Cys Gly Glu Glu 85 90 95 ArgArg Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100 105 110Ser Met Asn Thr Ala Ala Ile Ile Glu Gly 115 120 16 122 PRT ArtificialSequence Description of Artificial SequenceMurine IL5 modified bysubstitution with tetanus toxoid P2 epitope 16 Met Glu Ile Pro Met SerThr Val Val Lys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg AlaLeu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His LysAsn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile LeuLys Asp Gln Thr Val Arg Gly Gly Thr Val Met 50 55 60 Arg Leu Phe Gln AsnLeu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Glu Lys Lys GlnTyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr 85 90 95 Glu Leu Arg ThrArg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100 105 110 Ser Met AsnThr Ala Ala Ile Ile Glu Gly 115 120 17 124 PRT Artificial SequenceDescription of Artificial SequenceMurine IL5 modified by substitutionwith tetanus toxoid P2 epitope 17 Met Glu Ile Pro Met Ser Thr Val ValLys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His GlnLeu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asp GlnThr Val Arg Gly Gly Thr Val Met 50 55 60 Arg Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Glu Lys Lys Cys Gly Glu GluArg Arg Arg Thr Arg Gln Phe Leu Asp 85 90 95 Tyr Leu Gln Glu Phe Leu GlySer Met Asn Thr Gln Tyr Ile Lys Ala 100 105 110 Asn Ser Lys Phe Ile GlyIle Thr Glu Leu Glu Gly 115 120 18 130 PRT Artificial SequenceDescription of Artificial SequenceMurine IL5 modified by substitutionwith tetanus toxoid P30 epitope 18 Met Glu Ile Pro Met Ser Thr Val ValLys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His GlnLeu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asp GlnThr Val Arg Gly Gly Thr Val Met 50 55 60 Arg Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Glu Lys Lys Cys Phe Asn AsnPhe Thr Val Ser Phe Trp Leu Arg Val 85 90 95 Pro Lys Val Ser Ala Ser HisLeu Glu Arg Arg Thr Arg Gln Phe Leu 100 105 110 Asp Tyr Leu Gln Glu PheLeu Gly Ser Met Asn Thr Ala Ala Ile Ile 115 120 125 Glu Gly 130 19 122PRT Artificial Sequence Description of Artificial SequenceMurine IL5modified by substitution with tetanus toxoid P30 epitope 19 Met Glu IlePro Met Ser Thr Val Val Lys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser AlaHis Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Phe Asn Asn 20 25 30 Phe ThrVal Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His 35 40 45 Leu GluCys Ile Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys Asp 50 55 60 Gln ThrVal Arg Gly Gly Thr Val Met Arg Leu Phe Gln Asn Leu Ser 65 70 75 80 LeuIle Lys Lys Tyr Ile Asp Arg Gln Glu Lys Lys Cys Gly Glu Glu 85 90 95 ArgArg Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100 105 110Ser Met Asn Thr Ala Ala Ile Ile Glu Gly 115 120 20 128 PRT ArtificialSequence Description of Artificial SequenceMurine IL5 modified bysubstitution with tetanus toxoid P30 epitope 20 Met Glu Ile Pro Met SerThr Val Val Lys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser Ala His Arg AlaLeu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His LysAsn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile LeuLys Asp Gln Phe Asn Asn Phe Thr Val Ser Phe 50 55 60 Trp Leu Arg Val ProLys Val Ser Ala Ser His Leu Glu Val Met Arg 65 70 75 80 Leu Phe Gln AsnLeu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln Glu 85 90 95 Lys Lys Cys GlyGlu Glu Arg Arg Arg Thr Arg Gln Phe Leu Asp Tyr 100 105 110 Leu Gln GluPhe Leu Gly Ser Met Asn Thr Ala Ala Ile Ile Glu Gly 115 120 125 21 130PRT Artificial Sequence Description of Artificial SequenceMurine IL5modified by substitution with tetanus toxoid P30 epitope 21 Met Glu IlePro Met Ser Thr Val Val Lys Glu Thr Leu Ala Leu Leu 1 5 10 15 Ser AlaHis Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val ProThr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly LeuAsp Ile Leu Lys Asp Gln Thr Val Arg Gly Gly Thr Val Met 50 55 60 Arg LeuPhe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 GluLys Lys Cys Gly Glu Glu Arg Arg Arg Thr Arg Gln Phe Leu Asp 85 90 95 TyrLeu Gln Glu Phe Leu Gly Ser Met Asn Thr Phe Asn Asn Phe Thr 100 105 110Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu 115 120125 Glu Gly 130 22 139 PRT Artificial Sequence Description of ArtificialSequenceMurine IL5 modified by substitution with tetanus toxoid P2 andP30 epitopes 22 Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu AlaLeu Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr MetArg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly GluIle Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asp Gln Thr Val Arg Gly GlyThr Val Met 50 55 60 Arg Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr IleAsp Arg Gln 65 70 75 80 Glu Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys PheIle Gly Ile Thr 85 90 95 Glu Leu Arg Thr Arg Gln Phe Leu Asp Tyr Leu GlnGlu Phe Leu Gly 100 105 110 Ser Met Asn Thr Phe Asn Asn Phe Thr Val SerPhe Trp Leu Arg Val 115 120 125 Pro Lys Val Ser Ala Ser His Leu Glu GluGly 130 135 23 15 PRT Clostridium tetani 23 Gln Tyr Ile Lys Ala Asn SerLys Phe Ile Gly Ile Thr Glu Leu 1 5 10 15 24 21 PRT Clostridium tetani24 Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 510 15 Ala Ser His Leu Glu 20 25 45 DNA Artificial Sequence CDS (1)..(45)Description of Artificial sequence DNA encoding His tag derived fromDrosophila melanogaster 25 atg aaa cac caa cac caa cat caa cat caa catcaa cat caa caa 45 Met Lys His Gln His Gln His Gln His Gln His Gln HisGln Gln 1 5 10 15 26 15 PRT Artificial Sequence Description ofArtificial sequencederived from Drosophila melanogaster 26 Met Lys HisGln His Gln His Gln His Gln His Gln His Gln Gln 1 5 10 15 27 381 DNAArtificial Sequence Description of Artificial Sequence Human Il-5modified by substitution with tetanus toxoid epitope 27 atc ccc aca gaaatt ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro Thr Glu IlePro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctt tct actcat cga act ctg ctg ata gcc aat gag act ctc cgg 96 Leu Leu Ser Thr HisArg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 att cct gtt cct gtacat aaa aat cac caa ctg tgc act gaa gaa atc 144 Ile Pro Val Pro Val HisLys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 ttt cag gga ata ggc acactc gag agt caa act gtg caa ggg ggt act 192 Phe Gln Gly Ile Gly Thr LeuGlu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 gtg gaa aga cta ttc aaa aacttg tcc tta ata aag aaa tac atc gat 240 Val Glu Arg Leu Phe Lys Asn LeuSer Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 ggc caa aaa aaa aag tgt ggacag tac atc aag gcc aac tcc aag ttc 288 Gly Gln Lys Lys Lys Cys Gly GlnTyr Ile Lys Ala Asn Ser Lys Phe 85 90 95 atc ggc atc acc gag ctg aga gtaaac caa ttc cta gac tat ctg cag 336 Ile Gly Ile Thr Glu Leu Arg Val AsnGln Phe Leu Asp Tyr Leu Gln 100 105 110 gag ttt ctt ggt gta atg aac accgag tgg ata ata gaa agt tga 381 Glu Phe Leu Gly Val Met Asn Thr Glu TrpIle Ile Glu Ser 115 120 125 28 126 PRT Artificial Sequence Descriptionof Artificial Sequence Human Il-5 modified by substitution with tetanustoxoid epitope 28 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys GluThr Leu Ala 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala AsnGlu Thr Leu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu CysThr Glu Glu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr ValGln Gly Gly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile LysLys Tyr Ile Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Gln Tyr Ile LysAla Asn Ser Lys Phe 85 90 95 Ile Gly Ile Thr Glu Leu Arg Val Asn Gln PheLeu Asp Tyr Leu Gln 100 105 110 Glu Phe Leu Gly Val Met Asn Thr Glu TrpIle Ile Glu Ser 115 120 125 29 375 DNA Artificial Sequence Descriptionof Artificial Sequence Human Il-5 modified by substitution with tetanustoxoid epitope 29 atc ccc aca gaa att ccc aca agt gca ttg gtg aaa gagacc ttg gca 48 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu ThrLeu Ala 1 5 10 15 ctg ctt tct act cat cga act ctg ctg ata gcc aat gagact ctc ttc 96 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Phe 20 25 30 aac aac ttc acc gtg agc ttc tgg ctg cgc gtg cct aag gtgagc gcc 144 Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val SerAla 35 40 45 agc cac ctg gag tgc act gaa gaa atc ttt cag gga ata ggc acactc 192 Ser His Leu Glu Cys Thr Glu Glu Ile Phe Gln Gly Ile Gly Thr Leu50 55 60 gag agt caa act gtg caa ggg ggt act gtg gaa aga cta ttc aaa aac240 Glu Ser Gln Thr Val Gln Gly Gly Thr Val Glu Arg Leu Phe Lys Asn 6570 75 80 ttg tcc tta ata aag aaa tac atc gat ggc caa aaa aaa aag tgt gga288 Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly 8590 95 gaa gaa aga cgg aga gta aac caa ttc cta gac tat ctg cag gag ttt336 Glu Glu Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe 100105 110 ctt ggt gta atg aac acc gag tgg ata ata gaa agt tga 375 Leu GlyVal Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120 30 124 PRT ArtificialSequence Description of Artificial Sequence Human Il-5 modified bysubstitution with tetanus toxoid epitope 30 Ile Pro Thr Glu Ile Pro ThrSer Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr His ArgThr Leu Leu Ile Ala Asn Glu Thr Leu Phe 20 25 30 Asn Asn Phe Thr Val SerPhe Trp Leu Arg Val Pro Lys Val Ser Ala 35 40 45 Ser His Leu Glu Cys ThrGlu Glu Ile Phe Gln Gly Ile Gly Thr Leu 50 55 60 Glu Ser Gln Thr Val GlnGly Gly Thr Val Glu Arg Leu Phe Lys Asn 65 70 75 80 Leu Ser Leu Ile LysLys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly 85 90 95 Glu Glu Arg Arg ArgVal Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 Leu Gly Val MetAsn Thr Glu Trp Ile Ile Glu Ser 115 120 31 393 DNA Artificial SequenceDescription of Artificial Sequence Human Il-5 modified by substitutionwith tetanus toxoid epitope 31 atc ccc aca gaa att ccc aca agt gca ttggtg aaa gag acc ttg gca 48 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu ValLys Glu Thr Leu Ala 1 5 10 15 ctg ctt tct act cat cga act ctg ctg atagcc aat gag act ctc cgg 96 Leu Leu Ser Thr His Arg Thr Leu Leu Ile AlaAsn Glu Thr Leu Arg 20 25 30 att cct gtt cct gta cat aaa aat cac caa ctgtgc act gaa gaa atc 144 Ile Pro Val Pro Val His Lys Asn His Gln Leu CysThr Glu Glu Ile 35 40 45 ttt cag gga ata ggc aca ctc gag agt caa ttc aacaac ttc acc gtg 192 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Phe Asn AsnPhe Thr Val 50 55 60 agc ttc tgg ctg cgc gtg cct aag gtg agc gcc agc cacctg gag gtg 240 Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His LeuGlu Val 65 70 75 80 gaa aga cta ttc aaa aac ttg tcc tta ata aag aaa tacatc gat ggc 288 Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr IleAsp Gly 85 90 95 caa aaa aaa aag tgt gga gaa gaa aga cgg aga gta aac caattc cta 336 Gln Lys Lys Lys Cys Gly Glu Glu Arg Arg Arg Val Asn Gln PheLeu 100 105 110 gac tat ctg cag gag ttt ctt ggt gta atg aac acc gag tggata ata 384 Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr Glu Trp IleIle 115 120 125 gaa agt tga 393 Glu Ser 130 32 130 PRT ArtificialSequence Description of Artificial Sequence Human Il-5 modified bysubstitution with tetanus toxoid epitope 32 Ile Pro Thr Glu Ile Pro ThrSer Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr His ArgThr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile Pro Val Pro Val HisLys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Phe Asn Asn Phe Thr Val 50 55 60 Ser Phe Trp Leu Arg ValPro Lys Val Ser Ala Ser His Leu Glu Val 65 70 75 80 Glu Arg Leu Phe LysAsn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly 85 90 95 Gln Lys Lys Lys CysGly Glu Glu Arg Arg Arg Val Asn Gln Phe Leu 100 105 110 Asp Tyr Leu GlnGlu Phe Leu Gly Val Met Asn Thr Glu Trp Ile Ile 115 120 125 Glu Ser 13033 375 DNA Artificial Sequence Description of Artificial Sequence HumanIl-5 modified by substitution with tetanus toxoid epitope 33 atc ccc acagaa att ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro Thr GluIle Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctt tctact cat cga act ctg ctg ata gcc aat gag act ctc cgg 96 Leu Leu Ser ThrHis Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 att cct gtt cctgta cat aaa aat cac caa ctg tgc act gaa gaa atc 144 Ile Pro Val Pro ValHis Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 ttt cag gga ata ggcaca ctc gag agt caa cag tac atc aag gcc aac 192 Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Gln Tyr Ile Lys Ala Asn 50 55 60 tcc aag ttc atc ggc atcacc gag ctg gtg gaa aga cta ttc aaa aac 240 Ser Lys Phe Ile Gly Ile ThrGlu Leu Val Glu Arg Leu Phe Lys Asn 65 70 75 80 ttg tcc tta ata aag aaatac atc gat ggc caa aaa aaa aag tgt gga 288 Leu Ser Leu Ile Lys Lys TyrIle Asp Gly Gln Lys Lys Lys Cys Gly 85 90 95 gaa gaa aga cgg aga gta aaccaa ttc cta gac tat ctg cag gag ttt 336 Glu Glu Arg Arg Arg Val Asn GlnPhe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 ctt ggt gta atg aac acc gagtgg ata ata gaa agt tga 375 Leu Gly Val Met Asn Thr Glu Trp Ile Ile GluSer 115 120 34 124 PRT Artificial Sequence Description of ArtificialSequence Human Il-5 modified by substitution with tetanus toxoid epitope34 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 510 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 2025 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 3540 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Gln Tyr Ile Lys Ala Asn 5055 60 Ser Lys Phe Ile Gly Ile Thr Glu Leu Val Glu Arg Leu Phe Lys Asn 6570 75 80 Leu Ser Leu Ile Lys Lys Tyr Ile Asp Gly Gln Lys Lys Lys Cys Gly85 90 95 Glu Glu Arg Arg Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe100 105 110 Leu Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120 35357 DNA Artificial Sequence Description of Artificial Sequence HumanIl-5 modified by substitution with tetanus toxoid epitope 35 atc ccc acagaa att ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro Thr GluIle Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctt tctact cat cga act ctg ctg ata gcc aat gag act ctc cag 96 Leu Leu Ser ThrHis Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Gln 20 25 30 tac atc aag gccaac tcc aag ttc atc ggc atc acc gag ctg tgc act 144 Tyr Ile Lys Ala AsnSer Lys Phe Ile Gly Ile Thr Glu Leu Cys Thr 35 40 45 gaa gaa atc ttt caggga ata ggc aca ctc gag agt caa act gtg caa 192 Glu Glu Ile Phe Gln GlyIle Gly Thr Leu Glu Ser Gln Thr Val Gln 50 55 60 ggg ggt act gtg gaa agacta ttc aaa aac ttg tcc tta ata aag aaa 240 Gly Gly Thr Val Glu Arg LeuPhe Lys Asn Leu Ser Leu Ile Lys Lys 65 70 75 80 tac atc gat ggc caa aaaaaa aag tgt gga gaa gaa aga cgg aga gta 288 Tyr Ile Asp Gly Gln Lys LysLys Cys Gly Glu Glu Arg Arg Arg Val 85 90 95 aac caa ttc cta gac tat ctgcag gag ttt ctt ggt gta atg aac acc 336 Asn Gln Phe Leu Asp Tyr Leu GlnGlu Phe Leu Gly Val Met Asn Thr 100 105 110 gag tgg ata ata gaa agt tga357 Glu Trp Ile Ile Glu Ser 115 36 118 PRT Artificial SequenceDescription of Artificial Sequence Human Il-5 modified by substitutionwith tetanus toxoid epitope 36 Ile Pro Thr Glu Ile Pro Thr Ser Ala LeuVal Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu LeuIle Ala Asn Glu Thr Leu Gln 20 25 30 Tyr Ile Lys Ala Asn Ser Lys Phe IleGly Ile Thr Glu Leu Cys Thr 35 40 45 Glu Glu Ile Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Thr Val Gln 50 55 60 Gly Gly Thr Val Glu Arg Leu Phe LysAsn Leu Ser Leu Ile Lys Lys 65 70 75 80 Tyr Ile Asp Gly Gln Lys Lys LysCys Gly Glu Glu Arg Arg Arg Val 85 90 95 Asn Gln Phe Leu Asp Tyr Leu GlnGlu Phe Leu Gly Val Met Asn Thr 100 105 110 Glu Trp Ile Ile Glu Ser 11537 375 DNA Artificial Sequence Description of Artificial Sequence HumanIl-5 modified by substitution with tetanus toxoid epitope 37 atc ccc acagaa att ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro Thr GluIle Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctt tctact cat cga act ctg ctg ata gcc aat gag act ctc cgg 96 Leu Leu Ser ThrHis Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 att cct gtt cctgta cat aaa aat cac caa ctg tgc act gaa gaa atc 144 Ile Pro Val Pro ValHis Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 ttt cag gga ata ggcaca ctc gag agt caa act gtg caa ggg ggt act 192 Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 gtg gaa aga cta ttc aaaaac ttg tcc tta ata aag aaa tac atc gat 240 Val Glu Arg Leu Phe Lys AsnLeu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 ggc caa aaa aaa aag cagtac atc aag gcc aac tcc aag ttc atc ggc 288 Gly Gln Lys Lys Lys Gln TyrIle Lys Ala Asn Ser Lys Phe Ile Gly 85 90 95 atc acc gag ctg aga gta aaccaa ttc cta gac tat ctg cag gag ttt 336 Ile Thr Glu Leu Arg Val Asn GlnPhe Leu Asp Tyr Leu Gln Glu Phe 100 105 110 ctt ggt gta atg aac acc gagtgg ata ata gaa agt tga 375 Leu Gly Val Met Asn Thr Glu Trp Ile Ile GluSer 115 120 38 124 PRT Artificial Sequence Description of ArtificialSequence Human Il-5 modified by substitution with tetanus toxoid epitope38 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 510 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 2025 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 3540 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr 5055 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 6570 75 80 Gly Gln Lys Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly85 90 95 Ile Thr Glu Leu Arg Val Asn Gln Phe Leu Asp Tyr Leu Gln Glu Phe100 105 110 Leu Gly Val Met Asn Thr Glu Trp Ile Ile Glu Ser 115 120 39399 DNA Artificial Sequence Description of Artificial Sequence HumanIl-5 modified by substitution with tetanus toxoid epitope 39 atc ccc acagaa att ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro Thr GluIle Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctt tctact cat cga act ctg ctg ata gcc aat gag act ctc cgg 96 Leu Leu Ser ThrHis Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 att cct gtt cctgta cat aaa aat cac caa ctg tgc act gaa gaa atc 144 Ile Pro Val Pro ValHis Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 ttt cag gga ata ggcaca ctc gag agt caa act gtg caa ggg ggt act 192 Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 gtg gaa aga cta ttc aaaaac ttg tcc tta ata aag aaa tac atc gat 240 Val Glu Arg Leu Phe Lys AsnLeu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 ggc caa aaa aaa aag tgtgga ttc aac aac ttc acc gtg agc ttc tgg 288 Gly Gln Lys Lys Lys Cys GlyPhe Asn Asn Phe Thr Val Ser Phe Trp 85 90 95 ctg cgc gtg cct aag gtg agcgcc agc cac ctg gag aga gta aac caa 336 Leu Arg Val Pro Lys Val Ser AlaSer His Leu Glu Arg Val Asn Gln 100 105 110 ttc cta gac tat ctg cag gagttt ctt ggt gta atg aac acc gag tgg 384 Phe Leu Asp Tyr Leu Gln Glu PheLeu Gly Val Met Asn Thr Glu Trp 115 120 125 ata ata gaa agt tga 399 IleIle Glu Ser 130 40 132 PRT Artificial Sequence Description of ArtificialSequence Human Il-5 modified by substitution with tetanus toxoid epitope40 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 510 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 2025 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 3540 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr 5055 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 6570 75 80 Gly Gln Lys Lys Lys Cys Gly Phe Asn Asn Phe Thr Val Ser Phe Trp85 90 95 Leu Arg Val Pro Lys Val Ser Ala Ser His Leu Glu Arg Val Asn Gln100 105 110 Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr GluTrp 115 120 125 Ile Ile Glu Ser 130 41 393 DNA Artificial SequenceDescription of Artificial Sequence Human Il-5 modified by substitutionwith tetanus toxoid epitope 41 atc ccc aca gaa att ccc aca agt gca ttggtg aaa gag acc ttg gca 48 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu ValLys Glu Thr Leu Ala 1 5 10 15 ctg ctt tct act cat cga act ctg ctg atagcc aat gag act ctc cgg 96 Leu Leu Ser Thr His Arg Thr Leu Leu Ile AlaAsn Glu Thr Leu Arg 20 25 30 att cct gtt cct gta cat aaa aat cac caa ctgtgc act gaa gaa atc 144 Ile Pro Val Pro Val His Lys Asn His Gln Leu CysThr Glu Glu Ile 35 40 45 ttt cag gga ata ggc aca ctc gag agt caa act gtgcaa ggg ggt act 192 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val GlnGly Gly Thr 50 55 60 gtg gaa aga cta ttc aaa aac ttg tcc tta ata aag aaatac atc gat 240 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys TyrIle Asp 65 70 75 80 ggc caa aaa aaa aag ttc aac aac ttc acc gtg agc ttctgg ctg cgc 288 Gly Gln Lys Lys Lys Phe Asn Asn Phe Thr Val Ser Phe TrpLeu Arg 85 90 95 gtg cct aag gtg agc gcc agc cac ctg gag aga gta aac caattc cta 336 Val Pro Lys Val Ser Ala Ser His Leu Glu Arg Val Asn Gln PheLeu 100 105 110 gac tat ctg cag gag ttt ctt ggt gta atg aac acc gag tggata ata 384 Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Asn Thr Glu Trp IleIle 115 120 125 gaa agt tga 393 Glu Ser 130 42 130 PRT ArtificialSequence Description of Artificial Sequence Human Il-5 modified bysubstitution with tetanus toxoid epitope 42 Ile Pro Thr Glu Ile Pro ThrSer Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 Leu Leu Ser Thr His ArgThr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 Ile Pro Val Pro Val HisLys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 Phe Gln Gly Ile Gly ThrLeu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 Val Glu Arg Leu Phe LysAsn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 Gly Gln Lys Lys LysPhe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg 85 90 95 Val Pro Lys Val SerAla Ser His Leu Glu Arg Val Asn Gln Phe Leu 100 105 110 Asp Tyr Leu GlnGlu Phe Leu Gly Val Met Asn Thr Glu Trp Ile Ile 115 120 125 Glu Ser 13043 444 DNA Artificial Sequence Description of Artificial Sequence HumanIl-5 modified by substitution with tetanus toxoid epitopes 43 atc cccaca gaa att ccc aca agt gca ttg gtg aaa gag acc ttg gca 48 Ile Pro ThrGlu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr Leu Ala 1 5 10 15 ctg ctttct act cat cga act ctg ctg ata gcc aat gag act ctc cgg 96 Leu Leu SerThr His Arg Thr Leu Leu Ile Ala Asn Glu Thr Leu Arg 20 25 30 att cct gttcct gta cat aaa aat cac caa ctg tgc act gaa gaa atc 144 Ile Pro Val ProVal His Lys Asn His Gln Leu Cys Thr Glu Glu Ile 35 40 45 ttt cag gga ataggc aca ctc gag agt caa act gtg caa ggg ggt act 192 Phe Gln Gly Ile GlyThr Leu Glu Ser Gln Thr Val Gln Gly Gly Thr 50 55 60 gtg gaa aga cta ttcaaa aac ttg tcc tta ata aag aaa tac atc gat 240 Val Glu Arg Leu Phe LysAsn Leu Ser Leu Ile Lys Lys Tyr Ile Asp 65 70 75 80 ggc caa aaa aaa aagtgt gga cag tac atc aag gcc aac tcc aag ttc 288 Gly Gln Lys Lys Lys CysGly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 85 90 95 atc ggc atc acc gag ctgttc aac aac ttc acc gtg agc ttc tgg ctg 336 Ile Gly Ile Thr Glu Leu PheAsn Asn Phe Thr Val Ser Phe Trp Leu 100 105 110 cgc gtg cct aag gtg agcgcc agc cac ctg gag aga gta aac caa ttc 384 Arg Val Pro Lys Val Ser AlaSer His Leu Glu Arg Val Asn Gln Phe 115 120 125 cta gac tat ctg cag gagttt ctt ggt gta atg aac acc gag tgg ata 432 Leu Asp Tyr Leu Gln Glu PheLeu Gly Val Met Asn Thr Glu Trp Ile 130 135 140 ata gaa agt tga 444 IleGlu Ser 145 44 147 PRT Artificial Sequence Description of ArtificialSequence Human Il-5 modified by substitution with tetanus toxoidepitopes 44 Ile Pro Thr Glu Ile Pro Thr Ser Ala Leu Val Lys Glu Thr LeuAla 1 5 10 15 Leu Leu Ser Thr His Arg Thr Leu Leu Ile Ala Asn Glu ThrLeu Arg 20 25 30 Ile Pro Val Pro Val His Lys Asn His Gln Leu Cys Thr GluGlu Ile 35 40 45 Phe Gln Gly Ile Gly Thr Leu Glu Ser Gln Thr Val Gln GlyGly Thr 50 55 60 Val Glu Arg Leu Phe Lys Asn Leu Ser Leu Ile Lys Lys TyrIle Asp 65 70 75 80 Gly Gln Lys Lys Lys Cys Gly Gln Tyr Ile Lys Ala AsnSer Lys Phe 85 90 95 Ile Gly Ile Thr Glu Leu Phe Asn Asn Phe Thr Val SerPhe Trp Leu 100 105 110 Arg Val Pro Lys Val Ser Ala Ser His Leu Glu ArgVal Asn Gln Phe 115 120 125 Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val MetAsn Thr Glu Trp Ile 130 135 140 Ile Glu Ser 145 45 375 DNA ArtificialSequence Description of Artificial Sequence Murine Il-5 modified bysubstitution with tetanus toxoid epitope 45 atg gag att ccc atg agc acagtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro Met Ser Thr ValVal Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cga gct ctg ttgaca agc aat gag acg atg agg ctt cct 96 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Arg Leu Pro 20 25 30 gtc cct act cat aaa aat cac cagcta tgc att gga gag atc ttt cag 144 Val Pro Thr His Lys Asn His Gln LeuCys Ile Gly Glu Ile Phe Gln 35 40 45 ggg cta gac ata ctg aag aat caa actgtc cgt ggg ggt acc gtg gaa 192 Gly Leu Asp Ile Leu Lys Asn Gln Thr ValArg Gly Gly Thr Val Glu 50 55 60 atg cta ttc caa aac ctg tca tta ata aagaaa tac atc gat aga caa 240 Met Leu Phe Gln Asn Leu Ser Leu Ile Lys LysTyr Ile Asp Arg Gln 65 70 75 80 aaa gag aag tgt ggc cag tac atc aaa gctaac tcc aaa ttc atc ggt 288 Lys Glu Lys Cys Gly Gln Tyr Ile Lys Ala AsnSer Lys Phe Ile Gly 85 90 95 atc acc gag ctg agg acg agg cag ttc ctg gattat ctg cag gag ttc 336 Ile Thr Glu Leu Arg Thr Arg Gln Phe Leu Asp TyrLeu Gln Glu Phe 100 105 110 ctt ggt gtg atg agt aca gag tgg gca atg gaaggc taa 375 Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu Gly 115 120 46124 PRT Artificial Sequence Description of Artificial Sequence MurineIl-5 modified by substitution with tetanus toxoid epitope 46 Met Glu IlePro Met Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 Ser AlaHis Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val ProThr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly LeuAsp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 Met LeuPhe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 LysGlu Lys Cys Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 85 90 95 IleThr Glu Leu Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe 100 105 110Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu Gly 115 120 47 369 DNAArtificial Sequence Description of Artificial Sequence Murine Il-5modified by substitution with tetanus toxoid epitope 47 atg gag att cccatg agc aca gtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro MetSer Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cgagct ctg ttg aca agc aat gag acg atg ttc aac aac 96 Ser Ala His Arg AlaLeu Leu Thr Ser Asn Glu Thr Met Phe Asn Asn 20 25 30 ttc acc gtg agc ttctgg ctg cgc gtg ccc aag gtg agc gcc agc cac 144 Phe Thr Val Ser Phe TrpLeu Arg Val Pro Lys Val Ser Ala Ser His 35 40 45 ctg gag tgc att gga gagatc ttt cag ggg cta gac ata ctg aag aat 192 Leu Glu Cys Ile Gly Glu IlePhe Gln Gly Leu Asp Ile Leu Lys Asn 50 55 60 caa act gtc cgt ggg ggt accgtg gaa atg cta ttc caa aac ctg tca 240 Gln Thr Val Arg Gly Gly Thr ValGlu Met Leu Phe Gln Asn Leu Ser 65 70 75 80 tta ata aag aaa tac atc gataga caa aaa gag aag tgt ggc gag gag 288 Leu Ile Lys Lys Tyr Ile Asp ArgGln Lys Glu Lys Cys Gly Glu Glu 85 90 95 aga cgg agg acg agg cag ttc ctggat tat ctg cag gag ttc ctt ggt 336 Arg Arg Arg Thr Arg Gln Phe Leu AspTyr Leu Gln Glu Phe Leu Gly 100 105 110 gtg atg agt aca gag tgg gca atggaa ggc taa 369 Val Met Ser Thr Glu Trp Ala Met Glu Gly 115 120 48 122PRT Artificial Sequence Description of Artificial Sequence Murine Il-5modified by substitution with tetanus toxoid epitope 48 Met Glu Ile ProMet Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala HisArg Ala Leu Leu Thr Ser Asn Glu Thr Met Phe Asn Asn 20 25 30 Phe Thr ValSer Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser His 35 40 45 Leu Glu CysIle Gly Glu Ile Phe Gln Gly Leu Asp Ile Leu Lys Asn 50 55 60 Gln Thr ValArg Gly Gly Thr Val Glu Met Leu Phe Gln Asn Leu Ser 65 70 75 80 Leu IleLys Lys Tyr Ile Asp Arg Gln Lys Glu Lys Cys Gly Glu Glu 85 90 95 Arg ArgArg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100 105 110 ValMet Ser Thr Glu Trp Ala Met Glu Gly 115 120 49 387 DNA ArtificialSequence Description of Artificial Sequence Murine Il-5 modified bysubstitution with tetanus toxoid epitope 49 atg gag att ccc atg agc acagtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro Met Ser Thr ValVal Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cga gct ctg ttgaca agc aat gag acg atg agg ctt cct 96 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Arg Leu Pro 20 25 30 gtc cct act cat aaa aat cac cagcta tgc att gga gag atc ttt cag 144 Val Pro Thr His Lys Asn His Gln LeuCys Ile Gly Glu Ile Phe Gln 35 40 45 ggg cta gac ata ctg aag aat caa ttcaac aac ttc acc gtg agc ttc 192 Gly Leu Asp Ile Leu Lys Asn Gln Phe AsnAsn Phe Thr Val Ser Phe 50 55 60 tgg ctg cgc gtg ccc aag gtg agc gcc agccac ctg gag gtg gaa atg 240 Trp Leu Arg Val Pro Lys Val Ser Ala Ser HisLeu Glu Val Glu Met 65 70 75 80 cta ttc caa aac ctg tca tta ata aag aaatac atc gat aga caa aaa 288 Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys TyrIle Asp Arg Gln Lys 85 90 95 gag aag tgt ggc gag gag aga cgg agg acg aggcag ttc ctg gat tat 336 Glu Lys Cys Gly Glu Glu Arg Arg Arg Thr Arg GlnPhe Leu Asp Tyr 100 105 110 ctg cag gag ttc ctt ggt gtg atg agt aca gagtgg gca atg gaa ggc 384 Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu TrpAla Met Glu Gly 115 120 125 taa 387 50 128 PRT Artificial SequenceDescription of Artificial Sequence Murine Il-5 modified by substitutionwith tetanus toxoid epitope 50 Met Glu Ile Pro Met Ser Thr Val Val LysGlu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr SerAsn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln LeuCys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asn Gln PheAsn Asn Phe Thr Val Ser Phe 50 55 60 Trp Leu Arg Val Pro Lys Val Ser AlaSer His Leu Glu Val Glu Met 65 70 75 80 Leu Phe Gln Asn Leu Ser Leu IleLys Lys Tyr Ile Asp Arg Gln Lys 85 90 95 Glu Lys Cys Gly Glu Glu Arg ArgArg Thr Arg Gln Phe Leu Asp Tyr 100 105 110 Leu Gln Glu Phe Leu Gly ValMet Ser Thr Glu Trp Ala Met Glu Gly 115 120 125 51 351 DNA ArtificialSequence Description of Artificial Sequence Murine Il-5 modified bysubstitution with tetanus toxoid epitope 51 atg gag att ccc atg agc acagtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro Met Ser Thr ValVal Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cga gct ctg ttgaca agc aat gag acg atg cag tac atc 96 Ser Ala His Arg Ala Leu Leu ThrSer Asn Glu Thr Met Gln Tyr Ile 20 25 30 aaa gct aac tcc aaa ttc atc ggtatc acc gag ctg tgc att gga gag 144 Lys Ala Asn Ser Lys Phe Ile Gly IleThr Glu Leu Cys Ile Gly Glu 35 40 45 atc ttt cag ggg cta gac ata ctg aagaat caa act gtc cgt ggg ggt 192 Ile Phe Gln Gly Leu Asp Ile Leu Lys AsnGln Thr Val Arg Gly Gly 50 55 60 acc gtg gaa atg cta ttc caa aac ctg tcatta ata aag aaa tac atc 240 Thr Val Glu Met Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile 65 70 75 80 gat aga caa aaa gag aag tgt ggc gag gagaga cgg agg acg agg cag 288 Asp Arg Gln Lys Glu Lys Cys Gly Glu Glu ArgArg Arg Thr Arg Gln 85 90 95 ttc ctg gat tat ctg cag gag ttc ctt ggt gtgatg agt aca gag tgg 336 Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly Val MetSer Thr Glu Trp 100 105 110 gca atg gaa ggc taa 351 Ala Met Glu Gly 11552 116 PRT Artificial Sequence Description of Artificial Sequence MurineIl-5 modified by substitution with tetanus toxoid epitope 52 Met Glu IlePro Met Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 Ser AlaHis Arg Ala Leu Leu Thr Ser Asn Glu Thr Met Gln Tyr Ile 20 25 30 Lys AlaAsn Ser Lys Phe Ile Gly Ile Thr Glu Leu Cys Ile Gly Glu 35 40 45 Ile PheGln Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly 50 55 60 Thr ValGlu Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile 65 70 75 80 AspArg Gln Lys Glu Lys Cys Gly Glu Glu Arg Arg Arg Thr Arg Gln 85 90 95 PheLeu Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp 100 105 110Ala Met Glu Gly 115 53 369 DNA Artificial Sequence Description ofArtificial Sequence Murine Il-5 modified by substitution with tetanustoxoid epitope 53 atg gag att ccc atg agc aca gtg gtg aaa gag acc ttgaca cag ctg 48 Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu ThrGln Leu 1 5 10 15 tcc gct cac cga gct ctg ttg aca agc aat gag acg atgagg ctt cct 96 Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr Met ArgLeu Pro 20 25 30 gtc cct act cat aaa aat cac cag cta tgc att gga gag atcttt cag 144 Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile PheGln 35 40 45 ggg cta gac ata ctg aag aat caa act gtc cgt ggg ggt acc gtggaa 192 Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu50 55 60 atg cta ttc caa aac ctg tca tta ata aag aaa tac atc gat aga caa240 Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 6570 75 80 aaa gag aag cag tac atc aag gcc aac tcc aag ttc atc ggc atc acc288 Lys Glu Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr 8590 95 gag ctg agg acg agg cag ttc ctg gat tat ctg cag gag ttc ctt ggt336 Glu Leu Arg Thr Arg Gln Phe Leu Asp Tyr Leu Gln Glu Phe Leu Gly 100105 110 gtg atg agt aca gag tgg gca atg gaa ggc taa 369 Val Met Ser ThrGlu Trp Ala Met Glu Gly 115 120 54 122 PRT Artificial SequenceDescription of Artificial Sequence Murine Il-5 modified by substitutionwith tetanus toxoid epitope 54 Met Glu Ile Pro Met Ser Thr Val Val LysGlu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr SerAsn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln LeuCys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asn Gln ThrVal Arg Gly Gly Thr Val Glu 50 55 60 Met Leu Phe Gln Asn Leu Ser Leu IleLys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Lys Glu Lys Gln Tyr Ile Lys AlaAsn Ser Lys Phe Ile Gly Ile Thr 85 90 95 Glu Leu Arg Thr Arg Gln Phe LeuAsp Tyr Leu Gln Glu Phe Leu Gly 100 105 110 Val Met Ser Thr Glu Trp AlaMet Glu Gly 115 120 55 393 DNA Artificial Sequence Description ofArtificial Sequence Murine Il-5 modified by substitution with tetanustoxoid epitope 55 atg gag att ccc atg agc aca gtg gtg aaa gag acc ttgaca cag ctg 48 Met Glu Ile Pro Met Ser Thr Val Val Lys Glu Thr Leu ThrGln Leu 1 5 10 15 tcc gct cac cga gct ctg ttg aca agc aat gag acg atgagg ctt cct 96 Ser Ala His Arg Ala Leu Leu Thr Ser Asn Glu Thr Met ArgLeu Pro 20 25 30 gtc cct act cat aaa aat cac cag cta tgc att gga gag atcttt cag 144 Val Pro Thr His Lys Asn His Gln Leu Cys Ile Gly Glu Ile PheGln 35 40 45 ggg cta gac ata ctg aag aat caa act gtc cgt ggg ggt acc gtggaa 192 Gly Leu Asp Ile Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu50 55 60 atg cta ttc caa aac ctg tca tta ata aag aaa tac atc gat aga caa240 Met Leu Phe Gln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 6570 75 80 aaa gag aag tgt ggc ttc aac aac ttc acc gtg agc ttc tgg ctg cgc288 Lys Glu Lys Cys Gly Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg 8590 95 gtg ccc aag gtg agc gcc agc cac ctg gag agg acg agg cag ttc ctg336 Val Pro Lys Val Ser Ala Ser His Leu Glu Arg Thr Arg Gln Phe Leu 100105 110 gat tat ctg cag gag ttc ctt ggt gtg atg agt aca gag tgg gca atg384 Asp Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met 115120 125 gaa ggc taa 393 Glu Gly 130 56 130 PRT Artificial SequenceDescription of Artificial Sequence Murine Il-5 modified by substitutionwith tetanus toxoid epitope 56 Met Glu Ile Pro Met Ser Thr Val Val LysGlu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala His Arg Ala Leu Leu Thr SerAsn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys Asn His Gln LeuCys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu Lys Asn Gln ThrVal Arg Gly Gly Thr Val Glu 50 55 60 Met Leu Phe Gln Asn Leu Ser Leu IleLys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Lys Glu Lys Cys Gly Phe Asn AsnPhe Thr Val Ser Phe Trp Leu Arg 85 90 95 Val Pro Lys Val Ser Ala Ser HisLeu Glu Arg Thr Arg Gln Phe Leu 100 105 110 Asp Tyr Leu Gln Glu Phe LeuGly Val Met Ser Thr Glu Trp Ala Met 115 120 125 Glu Gly 130 57 387 DNAArtificial Sequence Description of Artificial Sequence Murine Il-5modified by substitution with tetanus toxoid epitope 57 atg gag att cccatg agc aca gtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro MetSer Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cgagct ctg ttg aca agc aat gag acg atg agg ctt cct 96 Ser Ala His Arg AlaLeu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 gtc cct act cat aaaaat cac cag cta tgc att gga gag atc ttt cag 144 Val Pro Thr His Lys AsnHis Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 ggg cta gac ata ctg aagaat caa act gtc cgt ggg ggt acc gtg gaa 192 Gly Leu Asp Ile Leu Lys AsnGln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 atg cta ttc caa aac ctg tcatta ata aag aaa tac atc gat aga caa 240 Met Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 aaa gag aag ttc aac aac ttcacc gtg agc ttc tgg ctg cgc gtg ccc 288 Lys Glu Lys Phe Asn Asn Phe ThrVal Ser Phe Trp Leu Arg Val Pro 85 90 95 aag gtg agc gcc agc cac ctg gagagg acg agg cag ttc ctg gat tat 336 Lys Val Ser Ala Ser His Leu Glu ArgThr Arg Gln Phe Leu Asp Tyr 100 105 110 ctg cag gag ttc ctt ggt gtg atgagt aca gag tgg gca atg gaa ggc 384 Leu Gln Glu Phe Leu Gly Val Met SerThr Glu Trp Ala Met Glu Gly 115 120 125 taa 387 58 128 PRT ArtificialSequence Description of Artificial Sequence Murine Il-5 modified bysubstitution with tetanus toxoid epitope 58 Met Glu Ile Pro Met Ser ThrVal Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala His Arg Ala LeuLeu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro Thr His Lys AsnHis Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu Asp Ile Leu LysAsn Gln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 Met Leu Phe Gln Asn LeuSer Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Lys Glu Lys Phe AsnAsn Phe Thr Val Ser Phe Trp Leu Arg Val Pro 85 90 95 Lys Val Ser Ala SerHis Leu Glu Arg Thr Arg Gln Phe Leu Asp Tyr 100 105 110 Leu Gln Glu PheLeu Gly Val Met Ser Thr Glu Trp Ala Met Glu Gly 115 120 125 59 438 DNAArtificial Sequence Description of Artificial Sequence Murine Il-5modified by substitution with tetanus toxoid epitopes 59 atg gag att cccatg agc aca gtg gtg aaa gag acc ttg aca cag ctg 48 Met Glu Ile Pro MetSer Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 tcc gct cac cgagct ctg ttg aca agc aat gag acg atg agg ctt cct 96 Ser Ala His Arg AlaLeu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 gtc cct act cat aaaaat cac cag cta tgc att gga gag atc ttt cag 144 Val Pro Thr His Lys AsnHis Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 ggg cta gac ata ctg aagaat caa act gtc cgt ggg ggt acc gtg gaa 192 Gly Leu Asp Ile Leu Lys AsnGln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 atg cta ttc caa aac ctg tcatta ata aag aaa tac atc gat aga caa 240 Met Leu Phe Gln Asn Leu Ser LeuIle Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 aaa gag aag tgt ggc cag tacatc aag gcc aac tcc aag ttc atc ggc 288 Lys Glu Lys Cys Gly Gln Tyr IleLys Ala Asn Ser Lys Phe Ile Gly 85 90 95 atc acc gag ctg ttc aac aac ttcacc gtg agc ttc tgg ctg cgc gtg 336 Ile Thr Glu Leu Phe Asn Asn Phe ThrVal Ser Phe Trp Leu Arg Val 100 105 110 ccc aag gtg agc gcc agc cac ctggag agg acg agg cag ttc ctg gat 384 Pro Lys Val Ser Ala Ser His Leu GluArg Thr Arg Gln Phe Leu Asp 115 120 125 tat ctg cag gag ttc ctt ggt gtgatg agt aca gag tgg gca atg gaa 432 Tyr Leu Gln Glu Phe Leu Gly Val MetSer Thr Glu Trp Ala Met Glu 130 135 140 ggc taa 438 Gly 145 60 145 PRTArtificial Sequence Description of Artificial Sequence Murine Il-5modified by substitution with tetanus toxoid epitopes 60 Met Glu Ile ProMet Ser Thr Val Val Lys Glu Thr Leu Thr Gln Leu 1 5 10 15 Ser Ala HisArg Ala Leu Leu Thr Ser Asn Glu Thr Met Arg Leu Pro 20 25 30 Val Pro ThrHis Lys Asn His Gln Leu Cys Ile Gly Glu Ile Phe Gln 35 40 45 Gly Leu AspIle Leu Lys Asn Gln Thr Val Arg Gly Gly Thr Val Glu 50 55 60 Met Leu PheGln Asn Leu Ser Leu Ile Lys Lys Tyr Ile Asp Arg Gln 65 70 75 80 Lys GluLys Cys Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 85 90 95 Ile ThrGlu Leu Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val 100 105 110 ProLys Val Ser Ala Ser His Leu Glu Arg Thr Arg Gln Phe Leu Asp 115 120 125Tyr Leu Gln Glu Phe Leu Gly Val Met Ser Thr Glu Trp Ala Met Glu 130 135140 Gly 145 61 57 DNA Homo sapiens CDS (1)..(57) DNA encoding naturalhuman IL5 leader sequence 61 atg agg atg ctt ctg cat ttg agt ttg ctg gctctt gga gct gcc tac 48 Met Arg Met Leu Leu His Leu Ser Leu Leu Ala LeuGly Ala Ala Tyr 1 5 10 15 gtg tat gcc 57 Val Tyr Ala 62 19 PRT Homosapiens 62 Met Arg Met Leu Leu His Leu Ser Leu Leu Ala Leu Gly Ala AlaTyr 1 5 10 15 Val Tyr Ala 63 60 DNA Mus musculus CDS (1)..(60) DNAencoding natural murine IL5 leader sequence 63 atg aga agg atg ctt ctgcac ttg agt gtt ctg act ctc agc tgt gtc 48 Met Arg Arg Met Leu Leu HisLeu Ser Val Leu Thr Leu Ser Cys Val 1 5 10 15 tgg gcc act gcc 60 Trp AlaThr Ala 20 64 20 PRT Mus musculus 64 Met Arg Arg Met Leu Leu His Leu SerVal Leu Thr Leu Ser Cys Val 1 5 10 15 Trp Ala Thr Ala 20 65 13 PRTArtificial Sequence Description of Artificial Sequence Promiscuous Thelper epitope derived from Homo sapiens 65 Ala Lys Phe Val Ala Ala TrpThr Leu Lys Ala Ala Ala 1 5 10

What is claimed is:
 1. A method for down-regulating interleukin 5 (IL5)activity in an anal in vivo, the method comprising: administering animmunogenically effective amount of at least one modified IL5polypeptide to the animal, wherein said modified IL5 polypeptidecomprises at least one modification in the IL5 polypeptide and whereinsaid modification comprises introduction of at least one foreign,immunodoninant T helper lymphocyte epitope (T_(H) epitope), results in aspecific reaction between the modified IL5 polypeptide and an antiserumraised against the animal's IL5 polypeptide, and is made in at least oneof loops 1-3 or in the amino acid residues C-terminal to helix D ofhuman and murine IL5, wherein the C-terminal of human IL5 corresponds toresidues 110-115 of SEQ ID No. 1, loop 1 of human IL5 corresponds toresidues 32-44 of SEQ ID NO. 1, loop 2 of human IL5 corresponds toresidues 58-64 of SEQ ID No. 1 and loop 3 of human IL5 corresponds toresidues 85-92 of SEQ ID NO. 1 and wherein the C-terminal of murine IL5corresponds to residues 108-113 of SEQ ID NO. 12, loop 1 of murine IL5corresponds to residues 30-42 of SEQ ID NO. 12, loop 2 of murine IL5corresponds to residues 56-62 of SEQ ID No. 12 and loop 3 of murine IL5corresponds to residues 83-90 of SEQ ID No. 12, whereby immunization ofthe animal with the modified IL5 polypeptide induces production ofantibodies against the animal's IL5 polypeptide.
 2. The method accordingto claim 1, wherein the modified IL5 polypeptide has a sequence identityof at least 70% with the animal's IL5 polypeptide.
 3. The methodaccording to claim 1, wherein said modification further comprises atleast one variation selected from the group consisting of at least onefirst moiety which effects targeting of the modified IL5 polypeptide toan antigen presenting cell (APC) or a B-lymphocyte, at least one secondmoiety, which stimulates the immune system, and at least one thirdmoiety, which optimizes presentation of the modified IL5 polypeptide tothe immune system.
 4. The method according to claim 3, wherein themodification comprises introduction as side groups, by binding to IL5,of the at least one variation, wherein the binding is selected from thegroup consisting of non-covalent and covalent binding.
 5. The methodaccording to claim 1, wherein the modification results in the productionof a fusion polypeptide.
 6. The method according to claim 1, wherein themodification results in the preservation of the overall tertiarystructure of IL5 as evidenced by the modified IL5 polypeptide reactingto the same extent as the IL5 molecule with a polyclonal antiseriumraised against the IL5 molecule.
 7. The method according to claim 1,wherein the modification comprises duplication of at least one IL5B-cell epitope.
 8. The method according to claim 1, wherein the at leastone foreign, immunodominant T-helper lymphocyte epitope is promiscuous.9. The method according to claim 8, wherein the at least one foreign,immunodominant T-helper lymphocyte epitope is selected from the groupcomprising a natural promiscuous T-helper lymphocyte epitope and anartificial MHC-II binding peptide sequence.
 10. The method according toclaim 9, wherein the natural T-helper lymphocyte epitope is selectedfrom a tetanus toxoid epitope, a diptheria toxoid epitope, an influenzavirus hemagluttinin epitope, and an epitope of P. falciparumcircumsporozoite (CS) protein.
 11. The method according to claim 3,wherein the first moiety is selected from the group consisting of asubstantially specific binding partner for a B-lymphocyte specificsurface antigen and a substantially specific binding partner for an APCspecific surface antigen.
 12. The method according to claim 3, whereinthe second moiety is selected from a cytokine, a hormone, and aheat-shock protein.
 13. The method according to claim 12, wherein thecytokine is selected from interferon γ(IFN-γ), fms like tyrosine kinase3L (Flt3L), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4(IL-4), interleukin 6 (IL-6), interleukin 12 (IL-12), interleukin 13(IL-13), interleukin 15 (IL-15), and granulocyte-macrophage colonystimulating factor (GM-CSF), and the heat-shock protein is selected fromheat shock protein 70 (HSP70), heat shock protein 90 (HSP90), heat shockcognate 70 (HSC70), glucose-regulated protein 94 (GRP94), andcalreticulin (CRT).
 14. The method according to claim 1, wherein the IL5polypeptide is a hunan IL5 polypeptide.
 15. The method according toclaim 14, wherein the human IL5 polypeptide has been modified bysubstituting at least one stretch of amino acids in SEQ ID NO: 1 with atleast one amino acid sequence of equal or different length therebygiving rise to a foreign T-helper lymphocyte (T_(H)) epitope, whereinsubstituted amino acid residues are selected from the group consistingof residues 87-90, residues 88-91, residues 32-43, residues 33-43,residues 59-64, residues 86-91, and residues 110-113 of SEQ ID NO: 1.16. The method according to claim 1, wherein presentation to the immunesystem is effected by having at least two copies of the modified IL5polypeptide linked to A carrier molecule that presents multiple copiesof antigenic determinants of the modified IL5 polypeptide.
 17. Themethod according to claim 1, wherein the modified IL5 polypeptide hasbeen formulated with an adjuvant.
 18. The method according to claim 17,wherein the adjuvant is selected from the group consisting of an immunetargeting adjuvant; a toxin; a cytokine; a mycobactexial derivative; anoil formulation; a polymer; a micelle forming adjuvant; a saponin, animmunostimulating complex matrix (an ISCOM matrix); a particle;dimethyldioctadecylammonium bromide, aluminium adjuvants; DNA adjuvants;γ-insulin; and an encapsulating adjuvant.
 19. The method according toclaim 1, wherein an effective amount of the modified IL5 polypeptide isadministered to the animal via a route selected from the parenteralroute; the peritoneal route; the oral route; the buccal route; thesublinqual route; the epidural route; the spinal route; the anal route;and the intracranial route.
 20. The method according to claim 19,wherein the effective amount is between 0.5 μg and 2,000 μg of themodified IL5 polypeptide.
 21. The method according to claim 19, whereinthe modified IL5 polypeptide is administered at least once a year. 22.The method according to claim 19, wherein the modified IL5 polypeptideis contained in a virtual lymph node (VLN) device.
 23. A method fortreatment, or amelioration of asthma or other chronic allergicconditions, the method comprising down-regulating IL5 activity accordingto the method of claim 1 whereby the number of eosinophil cells isreduced by at least 20%, either systemically or locally at the diseasefocus.
 24. The method according to claim 10, wherein the tetanus toxoidepitope is selected from the group consisting of P2 and P30.
 25. Themethod according to claim 11, wherein the first moiety is selected froma hapten and a carbohydrate for which there is a receptor on theB-lymphocyte or the antigen presenting cell (APC).
 26. The methodaccording to claim 3, wherein the third moiety is selected from thegroup consisting of a palmitoyl group, a mnyristyl group, a famesylgroup, a geranyl-geranyl group, a GPI-anchor, and an N-acyl diglyceridegroup.
 27. The method according to claim 7, wherein the modificationcomprises introduction of a hapten.
 28. The method according to claim 4,wherein the modification results in the production of a fusionpolypeptide.
 29. The method according to claim 1, wherein the animal isa human being.
 30. The method according to claim 19, wherein theparenteral route is selected from the group consisting of theintradermal route, the subdermal route, the intracutaneous route, thesubcutaneous route, and the intramuscular route.
 31. A modified IL5polypeptide which comprises at least one modification in the IL5polypeptide, whereby immunization of the animal with the modified IL5polypeptide induces production of antibodies against the animal's IL5polypeptide, wherein said modification comprises introduction of atleast one foreign, imnmunodominant T helper lymphocyte epitope (T_(H)epitope), results in a specific reaction between the modified IL5polypeptide and an antiserum raised against the animal's IL5polypeptide, and is made in at least one of loops 1-3 or in the aminoacid residues C-terminal to helix D, of human and murine IL5, whereinthe C-terninal of human IL5 corresponds to residues 110-115 of SEQ IDNo. 1, loop 1 of human IL5 corresponds to residues 32-44 of SEQ ID NO.1, loop 2 of human IL5 corresponds to residues 58-64 of SEQ ID No. 1 andloop 3 of human IL5 corresponds to residues 85-92 of SEQ ID NO. 1 andwherein the C-terminal of murine IL5 corresponds to residues 108-113 ofSEQ ID NO. 12, loop 1 of murine IL5 corresponds to residues 30-42 of SEQID NO. 12, loop 2 of murine IL5 corresponds to residues 56-62 of SEQ IDNo. 12 and loop 3 of murine IL 5 corresponds to residues 83-90 of SEQ IDNo.
 12. 32. An immunogenic composition comprising an immunogenicallyeffective amount of a modified IL5 polypeptide according to claim 31,the composition further comprising a pharmaceutically andimmunologically acceptable carrier or vehicle or combination thereof.33. The immunogenic composition according to claim 31, which furthercomprises an adjuvant.
 34. An immunogenic composition according to claim33, wherein the adjuvant is selected from the group consisting of animmune targeting adjuvant; a toxin; a cytokine; a mycobacterialderivative; an oil formulation; a polymer, a micelle forming adjuvant; asaponin; an immunostimulating complex matrix (an ISCOM matrix); aparticle; dimethyldioctadecylammonium bromide; aluminium adjuvants; DNAadjuvants; γ-inulin; and an encapsulating adjuvant.